CN101278059A - Method for diagnosing and treating renal cell carcinoma - Google Patents

Abstract

Objective methods for detecting and diagnosing renal cell carcinoma (RCC) are described herein. In one embodiment, the diagnostic method involves determining the expression level of RCC-associated gene that discriminates between RCC cells and normal cells. The present invention further provides methods of screening for therapeutic agents useful in the treatment of renal cell carcinoma, methods of treating renal cell carcinoma and method of vaccinating a subject against renal cell carcinoma.

Description

The method of diagnosis and treatment renal cell carcinoma

The application requires the right of U.S. Provisional Application serial number of submitting on July 28th, 2,005 60/703,640 and the U.S. Provisional Application serial number of submitting on May 11st, 2,006 60/799,960, and the full content of these applications is incorporated this paper into as a reference at this.

Technical field

The present invention relates to diagnose and treat the method for renal cell carcinoma.

Background technology

Renal cell carcinoma (RCC) is the 3rd common urogenital system malignant tumour, is all accounting for 2～3% in the human malignancies disease.At present, for limitation RCC tumour, surgical excision is the most effective treatment means, but for late period RCC patient do not have gratifying effective methods of treatment.Though it is about 20% that some RCC therapies related theretos efficient can reach, and severe side effect frequently occurs, and patient's prognosis not integral body improve (Ljungberg B, et al., (1999) B J U Int.84:405-11; LevyDA, et al. (1998) J Urol 159:1163-7.).The stage of tumour is considered to the informational prognostic factor of tool, and knows nothing basically about the molecular mechanism that the basis takes place as kidney.

The RCC tumour is based on that histological characteristic characterizes, such as the relevant cancer (cyst-associated carcinomas) of hyaline cell (80%), corpora mammillaria (papillary) (～10%), difficult dyeability (＜5%) or particulate state, spindle shape or tumour (5-15%).These histology hypotypes show unique clinical behavior separately, and wherein clear cell type and granule type often show and have more invasive clinical manifestation type.It is clear cell carcinoma that topmost type has been selected in this research.Particularly, the inventor has carried out large scale analysis to the gene expression pattern in such tumour.Some genes have been identified in the similar research of other research group's reports, and these genes may be useful (Takahashi M, et al. (2001) Proc Natl Acad Sci USA 98:9754-9 for the classification of prognosis purpose or RCC; Young AN, etal. (2001) Am J Pathol.158:1639-51; Skubitz KM ﹠amp; Skubitz AP. (2002) J LabClin Med 140:52-64; Takahashi M, et al. (2003) Oncogene 22:6810-8; HigginsJP, et al. (2003) Am J Pathol 162:925-32).By some research, found to be expected to become diagnostic markers or prognosis sign (profile) candidate's gene about the kidney gene expression pattern.But, can not reflect fully that from these data of tumor mass the expression in the kidney generating process changes, because the RCC cell generally exists with the form of the entity (solid mass) that contains the various kinds of cell component.What therefore, previous disclosed RCC expression pattern may reflect is inhomogenous pattern.

The cDNA microarray technology makes finds comprehensive gene expression pattern in normal cell and the malignant cell, and the genetic expression of comparing between malignant cell and corresponding normal cell becomes possible (Okabe etal., (2001) Cancer Res 61:2129-37; Kitahara et al., (2001) Cancer Res 61:3544-9; Lin et al., (2002) Oncogene 21:4120-8; Hasegawa et al., (2002) CancerRes 62:7012-7.).This means make people can illustrate the complicated character of cancer cells and understand the mechanism of oncogenesis.Be tested and appraised the gene that in tumour, takes off adjusting (deregulated), can diagnose various cancers more accurately, can also develop new treatment target (Bienz and Clevers, (2000) Cell103:311-20.).In order to separate the mechanism on bright tumour basis from the visual angle of genome integral body, and the target molecule of finding new medicine exploitation, diagnosis, we use the cDNA microarray of 23040 genes to resolve the expression pattern of some tumour cells (Okabe et al., (2001) Cancer Res 61:2129-37; Kitahara et al., (2001) Cancer Res 61:3544-9; Lin et al., (2002) Oncogene21:4120-8; Hasegawa et al., (2002) Cancer Res 62:7012-7.).

The research that is intended to disclose the cancer mechanism provides convenience for the molecular target of identifying some antineoplastic agent.For example, originally exploitation is used to suppress the farnesyl transferase inhibitor (FTI) of Ras relative growth signal transduction path (its activation depends on translation back farnesylation), be proved to be and in animal model, can have effectively treated Ras dependent tumors (Sun J etc., (1998) Oncogene 16:1467-73.).Similarly, use cancer therapy drug, be purpose with antagonism proto-oncogene acceptor HER2/neu, unite the clinical trial of using carcinostatic agent and anti-HER 2 monoclonal antibody trastuzumab that human body is carried out, patient with breast cancer's clinical response and overall existence (Molina MA have been improved, Deng, (2001) CancerRes.; 61 (12): 4744-9.).At last, developed a kind of tyrosine kinase inhibitor STI-571 of selectivity inactivation bcr-abl fusion rotein, be used for the treatment of the chronic myelogenous leukemia that the activation of bcr-abl Tyrosylprotein kinase composing type plays a crucial role in white corpuscle transforms.The medicinal design of these kinds is used to suppress the carcinogenic activity (O ' Dwyer ME﹠amp of specific gene product; Druker BJ. (2000) Curr OpinOncol.; 12 (6): 594-7).Therefore, the gene product that generally raises in cancer cells obviously can become the potential target of the new carcinostatic agent of exploitation.

Further verified, epitope peptide that present, that derive from tumor associated antigen (TAA) on CD8+ cytotoxic T cell (CTL) the identification I class MHC molecule, and cracking tumour cell.Since finding that the first TAA is MAGE family, adopted the immunology means to have been found that a lot of other TAA (Boon, (1993) Int J Cancer 54:177-80; Boon and van der Bruggen, (1996) J ExpMed 183:725-9; Van der Bruggen etc., (1991) Science 254:1643-7; Brichard V etc., (1993) J Exp Med 178:489-95; Kawakami Y etc., (1994) J Exp Med 180:347-52.).At present, some newfound TAA just accept clinical development as the target of immunotherapy.The TAA of Fa Xianing comprises MAGE (van der Bruggen etc. up to now, (1991) Science 254:1643-7), gp100 (Kawakami Y etc., (1994) J Exp Med 180:347-52), SART (Shichijo S etc., J Exp Med 187:277-88) and NY-ESO-1 (Chen YT etc., (1997) Proc Natl Acad Sci USA 94:1914-8) (1998).On the other hand, confirmed that in tumour cell specificity crosses the target that the gene product of high expression level (over-expressed) can be used as the inducing cell immunne response and be identified.Such gene product comprises p53 (Umano Y etc., (2001) HER2/neu (Tanaka H etc., (200 1) Brit J Cancer 84:94-9) Brit J Cancer 84:1052-7),, CEA (NukayaI etc., (1999) Int J Cancer 80:92-7) etc.

Although in basis relevant and clinical study, obtained marked improvement (RosenbergSA etc., (1998) Nature Med 4:321-7. with TAA; Mukherji B etc., (1995) Proc Natl Acad SciUSA 92:8078-82.; But existingly be used for the treatment of gland cancer for example the quantity of the candidate TAA of colorectal carcinoma is still very limited Hu X etc., (1996) Cancer Res 56:2479-83.).To be confined to the TAA in the cancer cells may be ideal candidate immunotherapy target for great expression and its expression in cancer cells.In addition, the evaluation of the novel TAA of powerful and specific anti-tumor immune response be can induce, clinical application (Boon and van der Bruggen, (1996) J ExpMed 183:725-9. are expected to promote at the peptide vaccination strategy of multiclass cancer; Van der Bruggen etc., (1991) Science 254:1643-7.; Brichard V etc., (1993) J Exp Med 178:489-95.; Kawakami Y etc., (1994) J Exp Med 180:347-52.; Shichijo S etc., (1998) J Exp Med 187:277-88.; Chen YT etc., (1997) Proc Natl Acad Sci USA 94:1914-8.; Harris CC, (1996) J Natl Cancer Inst 88:1442-5.; Butterfield LH etc., (1999) Cancer Res 59:3134-42.; Vissers JL etc., (1999) Cancer Res 59:5554-9.; Van der Burg SH etc., (1996) J Immunol 156:3308-14.; Tanaka F etc., (1997) Cancer Res 57:4465-8.; Fujie T etc., (1999) Int JCancer 80:169-72.; Kikuchi M etc., (1999) Int J Cancer 81:459-66.; Oiso M etc., (1999) Int J Cancer 81:387-94.).

According to report repeatedly, the peripheral blood mononuclear cell (PBMC) that stimulates through peptide that is derived from some healthy donors is made peptide and is replied and produce the IFN-γ of conspicuous level, but ⁵¹Cr-seldom brings into play cytotoxicity (Kawano K etc., (2000) Cancer Res 60:3550-8. with HLA-A24 or A0201 restrictive one to tumour cell in discharging and measuring; Nishizaka S etc., (2000) Cancer Res 60:4830-7.; Tamura M etc., (2001) Jpn J Cancer Res 92:762-7.).Yet with the same in the white race crowd, HLA-A24 and HLA-A0201 all are common HLA allelotrope (Date Y etc., (1996) Tissue Antigens 47:93-101. in the Japanese; Kondo A etc., (1995) J Immunol155:4307-12.; Kubo RT etc., (1994) J Immunol 152:3913-24.; Imanishi T etc., (1992) Proceeding of the eleventh International Histocompatibility Workshopand Conference Oxford University Press, Oxford, 1065.; Williams F etc., (1997) Tissue Antigen 49:129.).Therefore, the cancer antigen peptide of being presented by these HLA may be particularly useful for the treatment of the cancer among Japanese and the white people crowd.In addition, the peptide of known use high density usually can be at the CTL of external evoked low-affinity, go up at antigen presenting cell (APC) thus and produce high-caliber specific peptide/MHC mixture, effectively activate these CTL (Alexander-Miller MA etc., (1996) Proc Natl Acad Sci USA 93:4102-7.).

Therefore, carried out extensive parsing in order to be devoted to illustrate the relevant cancer mechanism of cancer and to identify the target of possible new carcinostatic agent exploitation, use the cDNA microarray of showing 27436 kinds of genes to the genetic expression in the kidney cancer cell colony of purifying is graphic.Particularly, unite and use cDNA microarray and laser beam micro-dissection, investigated the definite full genomic expression pattern of 15 routine samples of renal cell carcinoma.The discovery prompting of describing in detail in this specification sheets: these certified genes may play an important role in the growing multiplication of tumour cell, so they are targets likely of cancer therapy drug exploitation.

Summary of the invention

The present invention relates to the genetic expression graphic discovery relevant with renal cell carcinoma (RCC).In this manual, the gene of differential expression in the renal cell carcinoma is referred to as " RCC nucleic acid " or " RCC polynucleotide ", corresponding is called " RCC polypeptide " or " RCC albumen " by its encoded polypeptides.

In order to characterize the molecular mechanism that is associated with kidney, the cDNA microarray and the laser microbeam micro-dissection (LMM) of 27436 Human genomes showed in inventor's coupling, compare with normal renal cortex, analyzed the full genomic gene expression pattern of 15 routine renal cell carcinoma (RCC) surgery samples.The inventor has identified 251 general genes that raise in renal cell carcinoma, has also identified the gene of 721 general downward modulations in renal cell carcinoma.Through identify that it is unknown that the function of 74 up-regulated genes and 189 genes of reducing is arranged at present in renal cell carcinoma.The sxemiquantitative RT-PCR experimental result of 19 representative candidate genes has been supported the confidence level of the inventor's microarray analysis.These data provide the useful information of seeking candidate gene, and the product of these candidate genes may be the relevant molecular target of RCC treatment.

Thereby, the invention provides by determining to be derived from patient's biological sample, as RCC Expression of Related Genes level in the tissue sample, diagnose the renal cell carcinoma among the experimenter or determine that method of the proneness (predisposition) of renal cell carcinoma takes place for it.In this manual, term " RCC genes involved " is meant and it is characterized in that expression level differentiated gene between RCC cell and normal cell.Normal cell is the cell that derives from normal kidney tissue.In the context of the present invention, the RCC genes involved is one or more genes that are numbered in the gene of RCC 1-972.Compare with the normal control level of gene, the change of gene expression dose (for example rise or descend) represents that this experimenter suffers from RCC or has the danger that RCC takes place.

In the context of the present invention, term " control level " is meant the protein expression level that detects in control sample, and it comprises normal control level and RCC control level.Control level can be graphic from the single expression of single reference group, also can be graphic from the graphic single expression of a plurality of expression.The graphic database of expression of the cell that detects before for example, control level can derive from.The gene expression dose that " normal control level " is meant in the normal health individuality or detects in the group of the individuality of the known RCC of not suffering from.Normal individual is the individuality that does not have the renal cell carcinoma clinical symptom.On the other hand, " RCC control level " is meant the RCC Expression of Related Genes pattern of finding in suffering from the crowd of RCC.

Compared with the normal control level, in given the test agent, detect one or more gene expression doses that are numbered RCC 1-251 and rise, show that (obtaining sample by it) experimenter suffers from RCC or has the danger that RCC takes place.On the contrary, compared with the normal control level, in given the test agent, detect one or more gene expression doses that are numbered RCC252-972 and descend, show that this experimenter suffers from RCC or has the danger that RCC takes place.

Perhaps, a series of RCC Expression of Related Genes in the sample can be compared with the RCC control level of this serial genes.Similarity between sample is expressed and to be expressed with the RCC contrast shows the danger that (obtaining sample by it) experimenter suffers from RCC or has generation RCC.

According to the present invention, compare with control level when genetic expression and to rise or to descend 10%, 25% or 50% the time, can think that gene expression dose " changes ".In addition, compare with control level when genetic expression and to rise or to descend 1 times, 2 times, 5 times or when above, can think that gene expression dose changes.By detecting RCC genes involved probe and being derived from hybridization between patient's the genetic transcription thing of tissue sample, for example on array, determine genetic expression.

In the context of the present invention, the tissue sample that is derived from the patient is to derive from the experimenter, and for example known or suspection suffers from any tissue of the patient of RCC.For example, tissue can comprise epithelial cell.More specifically, tissue can be the epithelial cell from renal cell carcinoma (RCC).

The present invention also provides RCC reference table expression patterns, and it comprises plural gene expression dose among the RCC 1-972.Perhaps, RCC reference table expression patterns can also be by constituting more than two among the RCC 1-251 or among the RCC252-972.

The present invention also provides by making the subject cell of expressing the RCC genes involved and tried the activity that material contacted and measured RCC Expression of Related Genes level or activity or its gene product, identifying can suppress or strengthen RCC the genes involved for example expression of RCC 1-972 or the method for active working substance.Subject cell can be an epithelial cell, for example derives from the epithelial cell of renal cell carcinoma.Compare with the control level of this gene, the expression level of the RCC genes involved of rise (for example RCC 1-251) or the activity of its gene product reduce, and show that then this is tried the inhibitor that material is the RCC genes involved, can be used for alleviating the symptom of RCC.Perhaps, compare with the control level of this gene, the activity of the expression level of the RCC genes involved (for example RCC 252-972) of downward modulation or active or its gene product increases, and shows that then this is tried the toughener that material is RCC related gene expression or function, can be used for alleviating the symptom of RCC.

The present invention also provides test kit, and it contains and one or more RCC nucleic acid or RCC polypeptide bonded detection reagent.The present invention also provides and one or more RCC nucleic acid bonded nucleic acid arrays.

Methods of treatment of the present invention comprises the method for renal cell carcinoma among treatment or the prevention experimenter, and this method comprises the step of using the antisense composition to the experimenter.In the context of the present invention, the antisense composition reduces the particular target expression of gene.For example, the antisense composition can contain and the rise RCC genes involved sequence complementary Nucleotide that is selected from RCC 1-251.

Perhaps, method of the present invention can comprise the step of using siRNA (siRNA) composition to the experimenter.In the context of the present invention, the siRNA composition reduces the expression of nucleic acids that rise RCC genes involved for example is selected from RCC 1-251, especially reduces the expression of C6700V2 (RCC 81), B7032N (RCC 126) and B9320 (RCC 173).

In another method, treat or prevent renal cell carcinoma among the experimenter by use the ribozyme composition to the experimenter.Correspondingly, the present invention includes nucleic acid specificity ribozyme composition, said composition reduces the expression of raising RCC genes involved (for example RCC 1-251).Other methods of treatment comprises such method, wherein uses the expression that can improve one or more downward modulation RCC genes involveds (for example RCC252-972) to the experimenter, perhaps improves the active compound by one or more coded polypeptide in these genes.

The present invention also comprises vaccine and vaccine inoculation (vaccination) method.For example, the method for RCC can comprise to the experimenter and uses vaccine among treatment or the prevention experimenter, and described vaccine contains the immunologic competence fragment by the RCC polypeptide or the described polypeptide of the RCC nucleic acid encoding that is selected from RCC1-251.In the context of the present invention, the immunologic competence fragment is to be shorter in length than the total length native protein, but can induce the polypeptide with the similar immunne response of full-length proteins institute inductive immunne response.For example, the preferred segmental length of immunologic competence is at least 8 residues, and can the immune stimulatory cell, as T cell or B cell.Can produce by detection cell proliferation, cytokine (as IL-2) generation (elaboration) or antibody and measure the immunocyte stimulation.

The present invention also part based on following wonderful discovery: the expression that suppresses C6700, B7032N or B9320 is effective to the cell growth that suppresses to comprise the multiple cancer cells of renal cell carcinoma relevant cell in being grown in.The invention part of putting down in writing among the application is based on this discovery.

The present invention also provides cytostatic method.In these methods, the method that comprises the step that makes following composition and cells contacting also is provided, described composition comprises the siRNA (siRNA) that suppresses C6700, B7032N or B9320 expression.The present invention also provides the method that suppresses growth of tumour cell among the experimenter.These methods comprise the steps: to use the composition that contains siRNA (siRNA) to the experimenter, described siRNA and the sequence-specific hybridization that is selected from C6700, B7032N and B9320.Another aspect of the present invention provides the method for expression one or more in C6700, B7032N in the cell that suppresses biological sample or the B9320 gene.With double stranded RNA (RNA) molecule transfered cell, can suppress this expression of gene by the amount expressed with enough inhibition object genes (for example C6700, B7032N or B9320 gene).

Another aspect of the present invention relates to the product that comprises nucleotide sequence and carrier and comprises this product combination thing, and it is useful for for example implementing method of the present invention.Such siRNA molecule is provided in the product that provides, and when this siRNA molecule was imported the cell of expressing target gene, it suppressed one or more expression in C6700, B7032N or the B9320 gene.Also have such molecule in these molecules, they are made of sense strand and antisense strand, and described sense strand is the ribonucleoside acid sequence corresponding to C6700, B7032N or B9320 target sequence, and described antisense strand is and described sense strand complementary ribonucleoside acid sequence.The sense strand of this molecule and antisense strand mutual cross mutually form duplex molecule.

The present invention also provides cytostatic method.The composition and the cells contacting of the siRNA (siRNA) by making C6700, B7032N or B9320 can cell growth inhibiting.Cell is contacted with the transfection toughener.Cell can with in the body, external or stripped form provides.The experimenter is Mammals preferably, for example people, non-human primate, mouse, rat, dog, cat, horse or ox.Cell can be a for example nephrocyte of urogenital system.Perhaps, cell can be tumour cell (being cancer cell (cancer cell)), for example cancer cells (carcinoma cell) or adenocarcinoma cell.For example, cell can be renal cell carcinoma cell, more particularly hyaline cell.In the context of the present invention, phrase " cell growth inhibiting " refers to compare with untreated cell, the lower or viability decline of the rate of propagation of the cell that is subject to processing.Employing well known to a person skilled in the art that proliferation assay can measure cell growth.

The present invention further provides and newly transcribe variant C6700V2, it is not only the candidate diagnosis sign of renal cell carcinoma, still the target likely of exploitation diagnosis New Policy and effective antitumor agent.In preferred embodiment, the C6700V2 polypeptide comprises the albumen of 1151 amino acid (SEQ ID NO:66), and it is by the open reading frame coding of SEQ ID NO:65.

Except as otherwise noted, all technology used herein and scientific terminology all with the ordinary technical staff in the technical field of the invention conventional understand equivalent in meaning.Although implementing or check can be used when of the present invention and similar or the method and the material that are equal to described herein, the description below appropriate means and material such as this specification sheets.All publications that this paper mentions, patent application, patent and other reference are all incorporated this paper into as a reference in full.Existing under the situation of contradiction, be as the criterion with this specification sheets that comprises definition.In addition, material described herein, method and embodiment only are unrestricted the present invention for explanation.

An advantage of the method for this specification sheets record is to identify disease before detecting the obvious clinical symptom of renal cell carcinoma.By following detailed description and claims, other characteristics of the present invention and advantage are conspicuous.

Brief Description Of Drawings

Fig. 1 shows the photo for hyaline cell RCC (left drawing) and normal renal cortical cell (right drawing) use laser microbeam micro-dissection (LMM), before (a is capable) micro-dissection; After (b is capable) micro-dissection; (c is capable) is through the cell of micro-dissection.

Fig. 2 Fig. 2 (a) has shown the sxemiquantitative RT-PCR checking measurement result of highly expressing gene.By sxemiquantitative RT-PCR examination 21 genes (accession number .NM_018092, AA632745, NM_007250, W86513, BC077726, AA156409, W57613, CR749811, AW972553, AL832896, AK021778, AK026403, AK025204, NM_000677, AF070609, AI290343, AA442590, NM_000552, BC000234, BC034014 and NM_004567) and the expression of FDFT1 (internal contrast).The signal of microarray is corresponding with sxemiquantitative RT-PCR result of experiment.Normal renal cortex is to prepare from the 11 routine RCC patients that are used for this microarray.RPTEC represents kidney proximal tubule epithelial cell (renal proximal tubule epithelialcell).Fig. 2 (b) shows the expression of the C6700 in 11 RCC clones that adopt sxemiquantitative RT-PCR measurement.

C6700 expresses graphic Northern engram analysis result in Fig. 3 renal cell carcinoma clone and the health adult tissue.

Fig. 4 uses specific probe 3545 base transcripts in the multiple health adult tissue to be carried out result's (going up drawing) of multiple Northern engram analysis; Promptly use of the expression (following drawing) of the consensus sequence of 3545 base transcripts and 4533 base transcripts as F5749 in the probe analysis health adult tissue.

The Northern engram analysis result of C8919 transcript in Fig. 5 various human tissue.

The Northern engram analysis result (left drawing) of B7032N transcript in Fig. 6 various human tissue; RCC clone (Caki-1, Caki-2,786-O, A498, ACHN, 769-P, A704, RXF-631L, OS-RC-2, TUHR10TKB and TUHR14TKB), the Northern engram analysis result (right drawing) of B7032N transcript in RPTEC (kidney proximal tubule epithelial cell) and the normal people's organ (heart, lung, liver, kidney, testis and brain).

The Northern engram analysis result (left drawing) of B9320 transcript in Fig. 7 various human tissue; The Northern engram analysis result (right drawing) of B9320 transcript in RCC clone (Caki-1, Caki-2,786-O and A498), RPTEC (kidney proximal tubule epithelial cell) and the normal people's organ (heart, lung, liver, kidney, brain and testis).

The genome structure of Fig. 8 C6700.C6700 has 2 different variants, is called V1 and V2.Fig. 8 (a) has shown genome structure, and here, blank triangle is represented the existence of (inframe) terminator codon in the frame, and solid triangle is represented first methionine(Met).Fig. 8 (b) has shown the sxemiquantitative RT-PCR analytical results of SEMA5B, has wherein used from the RNA of 4 RCC clones (786-O, A704, OS-RC-2 and TUHR14TKB) and fetal brain poly A (+) RNA preparation.

The growth inhibitory effect of Fig. 9 siRNA (siRNA), described siRNA is designed to reduce the expression of C6700 in the RCC cell.Fig. 9 (a) sxemiquantitative RT-PCR result shows that the endogenous C6700 expression among the RCC clone OC-RC-2 is suppressed.β 2-MG is as internal contrast.The si-#2 carrier demonstrates and strikes low effect (knockdown effect); And the level that si-is out of order and si-simulates the C6700 transcript does not demonstrate any effect.With compare with si-simulation cells transfected with si-is out of order, carry out minimizing (Fig. 9 (c)) that transfection causes the colony number and the minimizing (Fig. 9 (b)) of survivaling cell number (is distinguished p＜0.005 and p＜0.001 with the si-#2 carrier; No paired t-test).

Proteic expression of exogenous B7032N and Subcellular Localization in Figure 10 COS7 cell.Figure 10 (a): transfection is after 24 hours and 48 hours, by the proteic exogenous expression of B7032N of Western trace demonstration; Figure 10 (b): the proteic Subcellular Localization of exogenous B7032 in the COS7 cell.

The siRNA of Figure 11 B7032N is to the low effect of striking of growth of RCC cell and cell survival.Figure 11 (a): the RXF-631L cell is to the effect of si-B7032N (#4) or contrast siRNA (si-is out of order), and by sxemiquantitative RT-PCR (upper left), colony forming assay (lower-left) and MTT measure (right side) and analyze.The level of B7032N transcript in the cell of handling with RT-PCR The effects siRNA.β 2-MG expression level is as quantitatively contrast.Si B7032N-#4 carrier demonstrates and strikes low effect; And the out of order level to the B7032N transcript of si-does not show any effect.With compare with the out of order cells transfected of si-, with the minimizing of B7032N-#4 carrier transfection causing colony number and minimizing (p＜0.0001 of viable count; No paired t-test).(b): the A498 cell is to the effect of si-B7032N (#4) or contrast siRNA (si-is out of order), and RT-PCR is upper left by sxemiquantitative), colony forming assay (lower-left) and MTT measure (right side) and analyze.The level of B7032N transcript in the cell of handling with RT-PCR The effects siRNA.β 2-MG expression level is as quantitatively contrast.The Si-B7032N-#4 carrier demonstrates and strikes low effect; And the out of order level to the B7032N transcript of si-does not show any effect.With compare with the out of order cells transfected of si-, with the minimizing of si-B7032N-#4 carrier transfection causing colony number and minimizing (p＜0.0001 of viable count; No paired t-test).

The growth effect of exogenous B7032N in Figure 12 NIH3T3 cell.Figure 12 (a): the cell of the exogenous B7032N of high level expression and with the Western engram analysis result of analog carrier cells transfected.Adopt anti-HA tag monoclonal antibody to confirm the exogenous importing that B7032N expresses.Beta-actin is as last sample contrast.Figure 11 (b): NIH3T3-B7032N cells in vitro growth.With the NIH3T3 cell of B7032N transfection (B7032N-A ,-B ,-C ,-D) and with the NIH3T3 cell of simulation transfection (simulation-A ,-B ,-C), measure by MTT.

Proteic expression of exogenous B9320 and Subcellular Localization in Figure 13 COS7 cell.Figure 12 (a): transfection is after 24 hours and 48 hours, the proteic exogenous expression of B9320 that the Western trace shows; Figure 13 (b): the proteic Subcellular Localization of exogenous B9320 in the COS7 cell.

The siRNA of Figure 14 B9320 is to the low effect of striking of growth of RCC cell and cell survival.Figure 14 (a): the Caki-2 cell is measured (right side) by sxemiquantitative RT-PCR (upper left), colony forming assay (lower-left) and MTT and is analyzed the effect of si-B9320 (#2) or contrast siRNA (si-is out of order).Test the level of B9320 transcript in the cell that investigation siRNA handled with RT-PCR.β 2-MG expression level is as quantitatively contrast.The Si-B9320-#2 carrier demonstrates and strikes low effect; And the out of order level to the B9320 transcript of si-does not show any effect.With compare with the out of order cells transfected of si-, with the minimizing of Si-B9320-#2 carrier transfection causing colony number and minimizing (p＜0.0001 of viable count; No paired t-test).Figure 14 (b): the A498 cell is measured (right side) by sxemiquantitative RT-PCR (upper left), colony forming assay (lower-left) and MTT and is analyzed the effect of Si-B9320-#2 or contrast siRNA (si-is out of order).The level of B9320 transcript in the cell of handling with RT-PCR The effects siRNA.β 2-MG expression level is as quantitatively contrast.The Si-B9320-#2 carrier demonstrates and strikes low effect; And the out of order level to the B9320 transcript of si-does not show any effect.With compare with the out of order cells transfected of si-, with the minimizing of Si-B9320-#2 carrier transfection causing colony number and minimizing (p＜0.0001 of viable count; No paired t-test).

The growth effect of exogenous B9320 in Figure 15 NIH3T3 cell.Figure 15 (a): the cell of the exogenous B9320 of high level expression and with the Western engram analysis result of analog carrier cells transfected.Adopt anti-HA tag monoclonal antibody to confirm the exogenous importing that B9320 expresses.Beta-actin is as last sample contrast.Figure 15 (b): NIH3T3-B9320 cells in vitro growth.With the NIH3T3 cell (B9320-1 ,-2 ,-3) of B9320 transfection with the NIH3T3 cell of simulating transfection (simulation-1 ,-2 ,-3), measure by MTT.

Detailed Description Of The Invention

Unless specific explanation is arranged in addition, the word that uses in this specification " (a) " or " a kind of (an) " or " should (the) " refer at least one or at least a.

Usually, using tumor mass (tumor mass) the RCC gene expression analysis that carries out of comparing with normal nephrocyte is distortion (distorted), this be because the RCC cell with the form existence of the entity (solid mass) that contains the various kinds of cell component. Like this, just comprise noise data (noisy data) among the result. Therefore, adopt a kind of method of separating pure cell colony-laser capture microdissection (LCM) method herein or claim laser microbeam microdissection (LMM) method, obtained specific cancer cell and normal epithelium cell (Kitahara et al., (200 1) Cancer Res, 61:3544-9; Gjerdrum et al., (2001) J Mol Diagn, 3:105-10.).

The present invention is partly based on following discovery: between the nephrocyte that derives from RCC patient and upper chrotoplast, the expression of multiple nucleic acids is graphic to change. The difference of gene expression is identified by comprehensive cDNA microarray system.

Use the cDNA microarray of dissecting 27648 genes of displaying that combine with laser capture microdissection, analyzed the gene expression pattern from the cancer cell of 15 routine RCC. By relatively adopt laser capture microdissection dissection method select purifying, from the expression pattern of the cancer cell of making a definite diagnosis the patient with normal nephrocyte, identified 251 genes and in the RCC cell, generally raised, the generally downward modulation in the RCC cell of 721 genes is arranged. Further, select possessing the candidate molecules sign that in patients serum or urine, detects the potentiality of cancer-associated protein, found the potential target of signal suppressing strategy among some exploit person RCC.

The difference expression gene of identifying in this specification has the diagnostic uses as RCC sign and RCC gene target, can change it and express to treat or alleviate the RCC symptom.

Expression is summarized among the table 4-5 by the gene of modulation (namely rise or descend) in RCC patient, in this specification it is referred to as " RCC phase correlation gene ", " RCC nucleic acid " or " RCC polynucleotides "; The corresponding polypeptide by its coding is called " RCC polypeptide " or " RCC albumen ". Unless otherwise indicated, " RCC " refers to the disclosed any sequence of this specification (for example RCC 1-972). For the gene of before having described, provide simultaneously its database login number.

By measuring the expression of range gene in the cell sample, can diagnose RCC. Similarly, measure these gene responses in the expression of various medicines, can identify the medicine that is used for the treatment of RCC.

The present invention includes shown in definite (for example measure) table 4-5 the expression of at least a in the sequence and even full sequence. Use is by the GenBank of known array^TMThe sequence information that the database login item provides can use technology for detection known to a person of ordinary skill in the art and measure mutually correlation gene of RCC. For example, can make up probe with the sequence corresponding to the RCC sequence in the entry of sequence data storehouse, be used for analyzing detection corresponding to the RNA sequence of RCC phase correlation gene at for example Northern blot hybridization. Probe typically comprises at least 10,20,50,100 or 200 nucleotides of canonical sequence. As another example, can use described sequence construct primer, be used for for example based on the detection method of amplification, as based on specific amplification RCC nucleic acid in the PCR of reverse transcription.

Then, with population of test cells, in the expression of organizing one or more RCC phase correlation genes in the sample that for example is derived from the patient and the reference group same base because of expression compare. Comprise that with reference to cell colony one or more are compared the known cell of parameter, i.e. nephrocyte cancer cell (for example RCC cell) or normal renal cortical cell (for example non-RCC cell).

Colony compares with the reference cell, and whether the gene expression pattern in the population of test cells can represent that RCC or its tendentiousness depend on the composition with reference to cell colony. For example, if formed by non-RCC cell with reference to cell colony, population of test cells and represent population of test cells right and wrong RCC with reference to the similitude of gene expression pattern between the cell colony. Otherwise, if formed by the RCC cell with reference to cell colony, population of test cells and represent that with reference to the similitude of the gene expression pattern between the cell colony population of test cells contains the RCC cell.

If in the population of test cells expression of RCC marker gene with differ by more than 1.0 times, 1.5 times, 2.0 times, 5.0 times or 10.0 times or more with reference to the expression of corresponding RCC marker gene in the cell colony, can think that it is changed.

Population of test cells and with reference to the differential gene expression between the cell colony can with respect to the contrast nucleic acid, for example housekeeping gene (housekeeping gene) carries out normalization. For example, contrast nucleic acid is the known nucleic acid that not there are differences because of cancer or the non-cancer state of cell. Can utilize the expression of contrast nucleic acid that the signal level in tested and the reference group is carried out normalization. Exemplary crt gene includes but not limited to for example beta-actin, glyceraldehyde 3 phosphate dehydrogenation enzyme and ribosomal protein P1.

Population of test cells can be compared with reference to cell colony with a plurality of. Wherein the known parameters of each reference group can be different. Therefore, population of test cells can be compared with reference to cell colony with reference to cell colony and known second of the non-RCC cell (normal cell) for example that contains with known first of the RCC cell for example that contains. Subject cell can be included in from the experimenter, known contain or the doubtful RCC of containing cell types of organization or cell sample in.

Subject cell is preferably available from bodily tissue or body fluid, for example biological fluid (such as blood, phlegm or urine). For example, subject cell can purifying from nephridial tissue. Preferably, population of test cells contains chrotoplast. Upper chrotoplast preferably from known or doubtful be the tissue of nephrocyte cancer.

With reference to the cell in the cell colony should from similar types of organization of the types of organization of subject cell. Optional ground is cell system with reference to cell colony, for example RCC cell system (i.e. positive contrast) or normal non-RCC cell system (i.e. negative contrast). Perhaps, control cells colony can be derived from the molecular information database, and described molecular information derives from tested parameter or the known cell of condition.

Preferred experimenter is mammal. Exemplary mammal includes but not limited to people, non-human primate, mouse, rat, dog, cat, horse or ox.

Can use methods known in the art, in the expression of albumen or the disclosed gene of nucleic acid level this specification of mensuration. For example, can use the Northern hybridization analysis to measure gene expression, described Northern hybridization analysis uses the probe of one or more sequences in these nucleotide sequences of specific recognition. In addition, can use for example specificity primer of difference expression gene sequence, adopt based on the PCR detection method of reverse transcription and measure gene expression. Can also measure at protein level and express, namely measure expression by measuring by polypeptide level or its biologically active of the described gene code of this specification. Such method is well known in the art, includes but not limited to for example immune determination method, and the method utilization is for the antibody of the albumen of described gene code. Biologically active by the albumen of these gene codes generally is known.

The term that uses in this specification " organism " refers to any live body of being made of a cell at least. Organism can be the unicellular so simple organism of eucaryon for example, also can be for example to comprise the people at the such complex biological body of interior mammal.

The term that uses in this specification " biological sample " refers to the subset (subset) of whole organism or its tissue, cell or component part (for example body fluid includes but not limited to blood, mucus, lymph liquid, synovia, celiolymph, saliva, amniotic fluid, Cord blood, urine, vaginal secretion and seminal fluid). Term " biological sample " also refers to from homogenate, the cracking product of the subset preparation of biological integral or its tissue, cell or component part, extracts thing, cell cultivation thing or tissue culture, or fraction wherein or a part. At last, " biological sample " refers to that also organism breeds the matrix of (propagate) therein, and for example nutrition culture medium or gel wherein contain the cell compositions such as albumen or nucleic acid molecules.

The diagnosis of nephrocyte cancer

In the context of the present invention, diagnose RCC by the expression of measuring one or more RCC nucleotide sequences in the population of test cells (namely being derived from patient's biological sample). Preferred population of test cells contains chrotoplast, the cell that is for example obtained by nephridial tissue. Can also from blood or other body fluid (for example urine), measure gene expression. Other biological sample also can be used for measuring protein level. For example, can adopt immune determination method or other conventional biology to measure from experimenter's to be diagnosed blood or the protein level in the serum.

Determine one or more RCC phase correlation genes in subject cell or the biological sample, for example expression of RCC 1-972, and specify the relevant normal control expression of RCC phase correlation gene to compare itself and tested one or more. The normal control level is the expression pattern that typically appears at the RCC phase correlation gene in the colony of the known RCC of not suffering from. Be derived from the organizing in the sample of patient, the variation of one or more RCC related gene expression levels (for example rise or descend) represents that this experimenter suffers from RCC or has the danger that RCC occurs. For example, compared with the normal control level, one or more raise the expression increase that the RCC gene is RCC 1-251 in the tested colony, represent that this experimenter suffers from RCC or has the danger that RCC occurs. Relative with it, compared with the normal control level, one or more down-regulated genes are the expression minimizing of RCC 252-972 in the tested colony, represent that this experimenter suffers from RCC or has the danger that RCC occurs.

Compared with the normal control level, the change of one or more RCC related gene expression levels represents that this experimenter suffers from RCC or has the danger that RCC takes place in the test population.For example, in a series of RCC genes involveds (RCC 1-251, RCC 252-972, RCC 1-972) at least 1%, at least 5%, at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or the change that more mostly occurs, represent that this experimenter suffers from RCC or has the danger that RCC takes place.

Corresponding to the mRNA of RCC genes involved or by the albumen of these genes encodings, can assess RCC Expression of Related Genes level in the biological sample by quantitative analysis.The quantitative analysis method of mRNA is well known to a person skilled in the art.For example, can assess level by Northern engram analysis or RT-PCR corresponding to the mRNA of RCC genes involved.The nucleotide sequence of RCC genes involved is known, and therefore, any those skilled in the art all can design probe or the primer that is used for quantitative analysis RCC genes involved.For example, exemplary C6700V2 Auele Specific Primer group is shown in the nucleotide sequence of SEQ IDNO:38 and 12.Perhaps, use and total nucleotide sequence annealed primer sets, can increase C6700V1 and C6700V2.When the amplified production of such primer sets comprised variant specific regions (being the disappearance of exon 2 1), amplified production may have nothing in common with each other aspect sequence length.For example, the primer of SEQ ID NO:83 and 84 nucleotide sequence can be used as primer sets and is used for two kinds of variants.And, can also use the specific probe of variant to detect amplified production.

In addition, these expression of gene levels can be based on being analyzed by RCC related gene coded proteic activity or amount (quantity).The method of measuring the related gene coded proteic amount of RCC has hereinafter been described.For example, can adopt immunoassay to determine proteic existence such in the biomaterial.As previously described, as long as express marker gene (being the RCC genes involved) in renal cell carcinoma patient's the sample, can be with any biomaterial as measuring albumen or its active biological sample.For example, uriniferous tubules is the example of suitable biological sample.And body fluid can be used for analyzing as blood and urine equally.On the other hand, in order to measure related gene coded proteic activity by RCC, can be according to the proteic active appropriate means of selecting to be analyzed.

RCC Expression of Related Genes level in the biological sample is assessed, and compared with the expression level in the normal specimens (for example being derived from non-ill experimenter's sample).If such expression of gene level that relatively shows than the expression level height in the normal specimens (the RCC genes involved shown in the table 4, i.e. 1-251) or low (the RCC genes involved shown in the table 5, i.e. 252-972), judges that then this experimenter suffers from RCC.Can measure simultaneously from normal subjects and waiting and diagnose RCC Expression of Related Genes level in experimenter's the biological sample.Perhaps can adopt statistical method,, determine the normal range of expression level according to the result that the analysis of gene expression dose the sample of gathering in advance from control group obtains.To compare from result and the normal range that experimenter's sample obtains, when this result does not fall into normal range, judge that then the experimenter suffers from RCC or has the danger that RCC takes place.

In the context of the present invention, also provide the diagnosis cell proliferative disease, as the diagnostic reagent of RCC.The example of diagnostic reagent of the present invention comprises polynucleotide or the polypeptide bonded compound with the RCC genes involved.Preferably, use the oligonucleotide with the multi-nucleotide hybrid of RCC genes involved, perhaps be used as such compound with the related gene coded polypeptide bonded antibody of RCC.RCC diagnostic method of the present invention can be used for estimating the validity of RCC treatment among the experimenter.According to present method, comprise the biological sample of population of test cells, be to obtain from the experimenter who accepts the RCC treatment.This evaluation method can be carried out according to the ordinary method of diagnosis RCC.

If wish, can be before treatment, in the treatment and/or the different time points after the treatment obtain biological sample by the experimenter.Measure RCC Expression of Related Genes level in the biological sample then, and with derive from the control level that for example comprises the known cell of RCC state (being cancer cells or non-cancer cells) and compare with reference to cell colony.Control level is not measured in accepting the biological sample of described treatment.

If control level is from the biological sample that does not contain cancer cells, the experimenter's who pays close attention to from the similarity of expression level in patient's the biological sample and control level treatment is effective so.And from the RCC related gene expression level in patient's the biological sample and the difference of control level, expression clinical consequences or prognosis are more undesirable.

Identify the working substance that suppresses or strengthen the RCC related gene expression

The population of test of expressing the relevant up-regulated gene of RCC is contacted with trying material, measure the activity of RCC Expression of Related Genes level or its gene product then, can identify the active material that suppresses RCC Expression of Related Genes or its gene product.Compare with control level (or not having expression or the activity level that is tried under the material conditions), exist the activity level of the RCC Expression of Related Genes level under the material conditions tried or its gene product to reduce, show that this material is the inhibitor of the relevant up-regulated gene of RCC, and may be useful in generation and the progress that suppresses RCC.

Perhaps, make the population of test of expressing the RCC genes involved and tried material and contact, determine the expression level of the down-regulated gene that RCC is relevant or the activity of its gene product then, can identify the expression or the active material of its gene product that strengthen the relevant down-regulated gene of RCC.Compare with the contrast expression level of RCC genes involved or activity (or not existing this to be tried level under the material conditions), exist the activity level of the RCC Expression of Related Genes level under the material conditions tried or its gene product to rise, show that this is tried material and can increase the expression of the relevant down-regulated gene of RCC or the activity of its gene product.

Population of test can be any cell of expressing the RCC genes involved.For example, population of test can comprise epithelial cell, as is derived from the cell of nephridial tissue.And subject cell can also be the immortalized cell line that is derived from adenocarcinoma cell.Perhaps, subject cell can also be with RCC genes involved cells transfected, or use with reporter gene can operate link to each other, from regulating and controlling sequence (for example promoter sequence) cells transfected of RCC genes involved.

Estimate the validity of RCC treatment among the experimenter:

The RCC genes involved of the differential expression of Jian Dinging in this manual, for the therapeutic process of monitoring RCC provides may.In the method, provide population of test cells by the experimenter who accepts the RCC treatment.In case of necessity, before treatment, the treatment in and/or the treatment after different time points obtain population of test cells by the experimenter.Then, measure one or more RCC Expression of Related Genes in the cell colony, and with comprise comparing of the known cell of RCC state with reference to cell colony.In the context of the present invention, should not accept described treatment with reference to cell colony.

If do not contain the RCC cell with reference to cell colony, population of test cells and the experimenter's that pays close attention to reference to the similarity of RCC related gene expression in the cell colony treatment is effective so.And population of test cells and normal control represent that with reference to the difference of RCC related gene expression in the cell colony clinical consequences or prognosis are more undesirable.Similarly, if contain the RCC cell with reference to cell colony, population of test cells and the experimenter's that pays close attention to reference to the difference between the RCC related gene expression in the cell colony treatment is effectively so, and population of test cells and normal control represent then that with reference to the similarity of RCC related gene expression in the cell colony clinical consequences or prognosis are more undesirable.

In addition, one or more RCC Expression of Related Genes levels (i.e. level before the treatment) of measuring in one or more RCC Expression of Related Genes levels (i.e. level after the treatment) measured in the biological sample that is derived from the experimenter that obtains after treatment and the biological sample that is derived from the experimenter that obtained before the treatment beginning can be compared.If the RCC genes involved is a up-regulated gene, the experimenter's that the minimizing of expression level is paid close attention in the sample of treatment back treatment is effectively so, and the increase of expression level or keep represents that then clinical consequences or prognosis are more undesirable in the sample of treatment back.On the contrary, if the RCC genes involved is a down-regulated gene, the experimenter's that the increase of expression level is paid close attention in the sample of treatment back treatment is effectively so, and the minimizing of expression level or keep represents that then clinical effectiveness or prognosis are more undesirable in the sample of treatment back.In this manual, term " effectively " expression is treated and is caused that the expression of gene that pathologic raises in the subject reduces, or the expression of gene increase of pathologic downward modulation, or the size of renal cell carcinoma, morbidity (prevalence) or metastatic potential reduce.When using described treatment, term " effectively " refers to treat the formation that postpones or prevent renal cell carcinoma, perhaps postpones, prevents or alleviate the clinical symptom of RCC when preventative.The evaluation of renal cell carcinoma can use the clinical procedures of standard to carry out.

In addition, can combine and differentiate validity with the method for any known diagnosis or treatment RCC.For example, by determining that symptomatic unusual (symptomatic anomalies) can carry out the diagnosis of RCC, describedly symptomaticly unusually for example lose weight, stomachache, backache, appetite stimulator, feel sick, vomiting and general malaise (generalized malaise), weakness and jaundice.

Selection is suitable for the concrete individual therapeutical agent that is used for the treatment of RCC:

The difference that genes of individuals is formed (genetic makeup) can cause the relative capacity of the multiple medicine of its metabolism difference to occur.Thereby play the working substance of anti-RCC agent effect at the subject intracellular metabolite, can the genetic expression of cancerous state characteristic is graphic to represent self to the graphic transformation of non-cancerous state characteristic genetic expression by inducing in experimenter's cell.Therefore, the RCC genes involved of the disclosed differential expression of this specification sheets, make and in from selected experimenter's population of test cells, test the RCC inhibitor with result of treatment or preventive effect of inferring, determine whether this material is that suitable R CC inhibitor becomes possibility in described experimenter.

In order to identify the RCC inhibitor that is suitable for concrete experimenter, will be exposed to from experimenter's population of test cells in the therapeutical agent, determine one or more RCC 1-972 expression of gene then.

In the context of the inventive method, population of test cells can contain the RCC cell of expressing the RCC genes involved.Preferred subject cell is an epithelial cell.For example, can have incubation population of test cells under the condition of candidate substances, the genetic expression of measuring given the test agent is graphic, and with one or more reference patterns, for example RCC reference table expression patterns or non-RCC reference table expression patterns compare.

With respect to contain RCC with reference to for the cell colony, one or more rises RCC gene is the expression decreased of RCC 1-251 in the population of test cells, perhaps one or more downward modulations RCC gene is the expression increase of RCC 252-972, represents that this material has result of treatment.

In the context of the present invention, being tried material can be any compound or composition.The exemplary material that tried that is fit to use in the present invention includes but not limited to immunomodulator.

Be used for the screening assay of identify therapeutic agents:

The RCC genes involved of the disclosed differential expression of this specification sheets also can be used to identify the candidate therapeutic agent of treatment RCC.Method of the present invention comprises the screening candidate therapeutic agent, whether can be to determine this candidate therapeutic agent with one or more RCC genes involveds, and it is graphic that for example the RCC status flag expression pattern of RCC 1-972 is transformed into the genetic expression of non-RCC status flag.

In the present invention, to be used for the treatment of for screening or to prevent the therapeutical agent of RCC be useful to RCC 1-972.

In the method, cellular exposure is tried material (successively or combination) in one or more, and measured the expression of one or more genes in described cell among the RCC 1-972.The RCC Expression of Related Genes pattern that to measure in test population is tried comparing with reference to identical RCC Expression of Related Genes level in the cell colony of material with not being exposed to.

Can stimulate the material that hangs down expression (under-expressed) expression of gene or suppressed the expression of cance high-expression gene to have the potential clinical benefit.Can verify further in animal or experimenter that these materials prevent that RCC from taking place and the ability of progress.

In another scheme, the invention provides the method for screening of candidate substances, described candidate substances is a target likely in the RCC treatment.As what above gone through, control the expression level of marker gene or the activity of their gene product, can control generation and the progress of RCC.Therefore, by adopting, can identify the candidate substances that is expected to become RCC treatment target with described expression level and active screening method as cancer or non-cancer state index.In the context of the present invention, described screening can comprise for example following step:

A) test-compound is contacted with polypeptide by the polynucleotide encoding that is selected from RCC 1-972;

B) detect the activity that combine between polypeptide and the test-compound; With

C) select and polypeptide bonded test-compound.

What be used to screen can be recombinant polypeptide by the marker gene encoded polypeptides, also can be to derive from natural albumen, or their partial peptide.With the contacted polypeptide of the present invention of test-compound for example can be purifying polypeptide, soluble proteins, with carrier-bound form or the fusion rotein that merges with other polypeptide.

As screening albumen, for example screen the proteic method of peptide bonded more than the marker gene coding, can use many methods that well known to a person skilled in the art.Such screening can be passed through, and for example, immuno-precipitation carries out particularly in the following manner.By marker gene being inserted the exogenous gene expression carrier, include but not limited to pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8 express marker gene in host (for example animal) cell etc.Being used for expression promoter can be normally used any promotor, comprise, for example, SV40 early promoter (Rigby in Williamson (ed.), (1982) Genetic Engineering, vol.3.Academic Press, London, 83-141), EF-α promotor (Kim et al., Gene 91:217-23 (1990)), CAG promotor (Niwa et al., (1991) Gene108:193-9), RSV LTR promotor (Cullen, (1987) Methods in Enzymology 152:684-704), SR α promotor (Takebe et al., (1988) Mol Cell Biol 8:466-72), CMV immediate early promoter (CMV immediate early promoter) (Seed and Aruffo, (1987) Proc Natl Acad Sci USA 84:3365-9), SV40 late promoter (Gheysen and Fiers, (1982) J Mol Appl Genet 1:385-94), gland virus stage starting (Adenovirus latepromoter) (Kaufman et al., (1989) Mol Cell Biol 9:946-58), HSV TK promotor etc.Can will desire expressed exogenous gene according to any method and import host cell, these methods are electroporation (Chu et al. for example, (1987) calcium phosphate method (Chenand Okayama Nucleic Acids Res 15:1311-26),, (1987) Mol Cell Biol 7:2745-52), deae dextran method (Lopata et al., (1984) Nucleic Acids Res 12:5707-17; Sussman and Milman, (1984) Mol CellBiol 4:1641-3), lipofection (Lipofectin) method (Derijard B, et al., (1994) Cell 76:1025-37; Lamb et al., (1993) Nature Genetics 5:22-30:Rabindran et al., (1993) Science 259:230-4) etc.By the recognition site (epi-position) of the known monoclonal antibody of specificity being imported the N-terminal or the C-terminal of polypeptide, can be with the formal representation of the fusion rotein of the recognition site that comprises monoclonal antibody by the marker gene encoded polypeptides.Also can use commercial available epitope-antibody system (Experimental Medicine 13:85-90 (1995)).Can utilize its multiple clone site expression is commercial available with for example carrier of the fusion rotein of beta-galactosidase enzymes, maltose-conjugated protein, glutathione-S-transferase, green fluorescent protein (GFP) etc.

Also reported the little epi-position of forming to tens amino acid by several by only introducing, made the characteristic of described polypeptide not because of fusion changes, and the fusion rotein of preparation.As polyhistidyl (His label), influenza virus agglutinator (influenza aggregate) HA, people c-myc, FLAG, vesicular stomatitis virus glycoprotein (Vesicular stomatitis virus glycoprotein) (VSV-GP), T7 gene 10 albumen (T7 label), human herpes simplex vicus's glycoprotein (human simple herpes virus glycoprotein) (HSV label), E label (epi-position on the mono-clonal phage) etc., and the monoclonal antibody of discerning them can screen as the epitope-antibody system can be in conjunction with the albumen (Experimental Medicine 13:85-90 (1995)) by the marker gene encoded polypeptides.

In immuno-precipitation, in the cell lysate of suitable washing composition (detergent) preparation, add these antibody, form immunocomplex.Described immunocomplex by the marker gene encoded polypeptide, have with the polypeptide and the antibody of this polypeptide bonded ability and form.Except that the antibody that uses anti-above-mentioned epi-position, can also use the antibody of anti-marker gene coded polypeptide to carry out immunoprecipitation, described antibody is preparation as mentioned above alternatively.

When described antibody is a kind of mouse IgG antibody, can be by for example albumin A Sepharose or Protein G Sepharose precipitation immunocomplex.If will be prepared into the fusion rotein that has epi-position such as GST by the marker gene encoded polypeptides, can use and these epitope specificity bonded materials, for example gsh-Sepharose 4B etc. forms immunocomplex according to the mode identical with the antibody that uses anti-polypeptide.

Immunoprecipitation can according to or abide by, for example the method in the document (Harlow and Lane, (1988) Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York) is carried out.

Usually use SDS-PAGE that the albumen of immunoprecipitation is analyzed, can use the gel of suitable concn to analyze bonded albumen by proteic molecular weight.Owing to adopt dyeing of normal dyeing rule such as coomassie (Coomassie) or silver dyeing to be difficult to detect and marker gene encoded polypeptide bonded albumen, thereby can be by the described proteic detection sensitivity of following method improvement: radio isotope contained ³⁵The S-methionine(Met) or ³⁵Culturing cell in the substratum of S-halfcystine, marker protein in cell, and detect albumen.When the molecular weight of known protein, can from the SDS-polyacrylamide gel, go out target protein and measure its sequence by direct purification.

As the proteic screening method of peptide bonded more than the marker gene coding, can adopt for example West-Western engram analysis (Skolnik et al, (1991) Cell 65:83-90).Specifically, can obtain by following method can be in conjunction with the albumen of marker gene encoded polypeptide: use phage vector (for example ZAP), by (for example expecting expression and the proteic cell of marker gene encoded polypeptide bonded, tissue, organ, tissue is as testis or ovary) or cultured cells (Caki-1 for example, Caki-2,786-O, A704, OS-RC-2, TUHR14TKB and A498) preparation cDNA library; Go up this albumen of expression at LB-agarose (LB-agarose), expressed proteins is fixed on the filter membrane (filter), make the polypeptide and the reaction of above-mentioned filter membrane of purified and mark again, express and the proteic plaque of marker gene encoded polypeptide bonded according to marker detection then.Can utilize combining between vitamin H and the avidin, perhaps can utilize combine with marker gene encoded polypeptides specificity or with peptide that is blended in this polypeptide or polypeptide (for example GST) specificity bonded antibody, to carrying out mark by the marker gene encoded polypeptides.Also can adopt the method for using radio isotope or fluorescence etc.

Perhaps, in another embodiment of screening method of the present invention, (" MATCHMAKER two-hybrid system ", " test kit is measured in Mammals MATCHMAKER double cross ", " MATCHMAKER single crosses system " are (Clontech) can to use the two-hybrid system (two-hybrid system) that utilizes cell; " HybriZAP double cross carrier system " (Stratagene); With reference to " Dalton andTreisman, (1992) Cell 68:597-612 ", " Fields and Sternglanz, (1994) TrendsGenet 10:286-92 ").

In two-hybrid system,, and in yeast cell, express polypeptide of the present invention and SRF-land or the fusion of GAL4-land.Express and the proteic cell preparation cDNA of polypeptide bonded of the present invention library by expection, make this library when expressing with VP16 or GAL4 transcription activating zone (transcriptional activation region) fusion.Then the cDNA library is imported above-mentioned yeast cell, and isolate the cDNA that is derived from described library (when yeast cell, expressing can be with polypeptide bonded albumen of the present invention the time from detected positive colony, both combinations activate reporter gene, make positive colony to be detected).By above-mentioned isolating cDNA is imported intestinal bacteria and expressing protein, can prepare by this cDNA encoded protein.

The example of reporter gene except that the HIS3 gene, also includes but not limited to Ade2 gene, lacZ gene, CAT gene, luciferase genes etc.

Adopt affinity chromatography can screen equally can with by marker gene encoded polypeptides bonded compound.For example, can will be fixed on the carrier of affinity column, and test-compound is added on this chromatography column by the marker gene encoded polypeptides, described test-compound comprise can with by marker gene encoded polypeptides bonded albumen.Test-compound in this specification sheets can be for example cell extract, cell lysate etc.Behind the sample, chromatography column is washed on described test-compound, can prepare and described polypeptide bonded compound.

When test-compound is albumen, analyze the proteic aminoacid sequence that obtains, based on the synthetic oligo DNA of this sequence, be probe screening cDNA library, thereby obtain the DNA of encoding said proteins with this oligo DNA.

In the present invention, can will utilize the biosensor (biosensor) of surface plasma resonance (surface plasmonresonance phenomenon) the bonded compound to be detected or quantitative means with work.When using this class biosensor, only use the polypeptide of minute quantity and do not need mark (BIAcore for example, Pharmacia), can be by the interaction between surface plasma body resonant vibration signal Real Time Observation polypeptide of the present invention and the test-compound.Therefore, it is possible using biosensor such as BIAcore to estimate by the combination between marker gene encoded polypeptides and the test-compound.

When immobilized when being exposed to synthetic compound, crude substance storehouse or random phage peptide display libraries by the marker gene encoded polypeptides, screen the method for bonded molecule with it, and be used to separate described albumen and in conjunction with described proteic compound (comprising agonist and antagonist), based on high-throughput screening method (Wrighton etc., (1996) Science 273:458-64 of combinatorial chemistry technique; Verdine, (1996) Nature 384:11-13), be well known to a person skilled in the art.

Perhaps, the polypeptide that the invention provides the service marking genes encoding screens the method that is used for the treatment of or prevents the compound of RCC, and this method may further comprise the steps:

(a) test-compound is contacted with polypeptide, described polypeptide is by the polynucleotide encoding that is selected from RCC 1-972;

(b) biological activity of the polypeptide of detection step (a); With

(c) select following compound: compare with the biological activity that detects under the condition that does not have test-compound, described compound suppresses the biological activity by the polypeptide of the polynucleotide encoding that is selected from RCC 1-251, or strengthens the biological activity by the polypeptide of the polynucleotide encoding that is selected from RCC 252-972.

Nucleotide sequence that can the service marking gene obtains can be used for the albumen of screening method of the present invention with the form of recombinant protein.Any polypeptide all can be used for screening, as long as they keep the biological activity by the marker gene encoded polypeptides.Based on the information relevant with marker gene and encoded protein thereof, those skilled in the art can select proteic any biological activity as screening index, and can select any suitable measuring method to measure the biological activity of selection.

Compound by this screening and separating is by the agonist of marker gene encoded polypeptides or the material standed for of antagonist.Term " agonist " refers to by combining the molecule that activates its function with described polypeptide.Similarly, term " antagonist " refers to by combining the molecule that suppresses its function with described polypeptide.And the isolated compound by this screening is the interior interactional candidate inhibitor of body or the stimulant of marker gene coded polypeptide and molecule (comprising DNA and albumen).

When the biological activity that detects in the method for the present invention is cell proliferation, can be by detecting: for example as the described following method of embodiment, the cell by the marker gene encoded polypeptides is expressed in preparation, there is the described cell of cultivation under the condition of test-compound, measure cell proliferation rate, measure the cell cycle etc., and the mensuration colony forms activity.

In another embodiment, the invention provides the method that screening is used for the treatment of or prevents the compound of RCC.As above describe in detail,, can control generation and the progress of RCC by the expression level of control marker gene.Thereby, be that index is screened by expression level with marker gene, can identify the compound that can be used for treating or preventing RCC.In the context of the present invention, such screening can comprise for example following steps:

(a) make candidate compound and the cells contacting of expressing one or more marker gene, wherein said one or more marker gene are selected from RCC 1-972; With

(b) select such candidate compound: its reduction is selected from the expression level of one or more marker gene of RCC 1-251, or improves the expression level of one or more marker gene that are selected from RCC 252-972.

Express the cell of marker gene, comprise the clone of for example setting up by RCC; Such cell can be used for above-mentioned screening of the present invention (for example Caki-1, Caki-2,786-O, A704, OS-RC-2, TUHR14TKB and A498).Expression level can adopt method well known to those skilled in the art to assess.In this screening method, the compound that reduces or strengthen the expression level of one or more marker gene is the candidate substances that is used for RCC treatment or prevention.

Perhaps, screening method of the present invention can may further comprise the steps:

A) make candidate compound and the cells contacting that has wherein imported carrier, wherein said carrier contains the transcription regulatory region of one or more marker gene and the reporter gene of expressing under the regulation and control of this transcription regulatory region, wherein said one or more marker gene are selected from RCC 1-972;

B) measure this report expression of gene level or activity; With

C) select such candidate compound: detected expression level is not compared during this candidate compound, and when described marker gene is when being selected from the rise marker gene of RCC 1-251, this compound reduces this report expression of gene or activity level; Or when described marker gene is when being selected from the downward modulation marker gene of RCC 252-972, this compound increases this report expression of gene level.

Suitable reporter gene and host cell are well known in the art.Service marking gene transcription control region can prepare the reporter constructs that is applicable to screening method of the present invention.When those skilled in the art's known markers gene transcription control region, can use previous sequence information to prepare reporter constructs.When the transcription regulatory region of marker gene is not identified yet, can from genomic library, separate the Nucleotide section that contains transcription regulatory region based on the nucleotide sequence information of marker gene.

The example that can be used for protein-bonded upholder (support) comprises insoluble polysaccharide, as agarose, Mierocrystalline cellulose and dextran; And synthetic resins, as polyacrylamide, polystyrene and silicon (silicon); Can preferably use by the commercial available pearl of above-mentioned materials preparation and plate (for example porous plate, biologic sensor chip etc.).When using pearl, they can be inserted pillar.

Albumen can carry out according to conventional methods with combining of upholder, and these methods are Chemical bond or physical adsorption for example.In addition, albumen can combine with upholder by its antibody of specific recognition.In addition, can also utilize avidin and biological usually implement combining of polypeptide and upholder.

Combination between the albumen is preferably carried out in damping fluid, and the example of damping fluid includes but not limited to phosphate buffered saline buffer and Tris damping fluid.But, any not between the arrestin bonded damping fluid all be suitable for.

Among the present invention, utilize the biosensor of surface plasma resonance can be used as to detect or the quantitative proteic device of bonded.(for example BIAcore in the time of Pharmacia), only uses a spot of polypeptide, and need not mark, can be by the interaction between the surface plasma body resonant vibration signal Real Time Observation albumen to use such biosensor.

Perhaps, can carry out mark to the marker gene encoded polypeptides, protein-bonded mark can be used for detecting or measuring bonded albumen.Particularly, a kind of albumen is carried out after the preliminary making, under the condition that test-compound exists, the albumen of mark is contacted with other albumen, then after washing according to marker detection or measure bonded albumen.

In the method for the present invention, can come labelled protein by the applying marking material, described mark substance such as radio isotope are (for example ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I), enzyme (for example alkaline phosphatase, horseradish peroxidase, beta-galactosidase enzymes, beta-glucosidase enzyme), fluorescent substance (for example, fluorescein isothiocyanate (FITC), rhodamine) and vitamin H/avidin.When with labelled with radioisotope albumen, can detect or measure by liquid flashing (liquid scintillation).Perhaps, by adding the substrate of this enzyme, the enzymatic that detects or measure substrate with absorption spectrophotometry (absorptiometer) changes (as producing color), thereby can detect or measure the albumen with enzyme labelling.In addition, with in the situation of marking, can utilize fluorophotometer to detect or measure bonded albumen fluorescent substance.

When in the linguistic context of screening method of the present invention, using antibody, preferably utilize one of above-mentioned mark substance that described antibody is carried out mark, and detect or measure based on mark substance.Perhaps, also can use second antibody that it is detected with resisting the antibody of marker gene encoded polypeptides or vitamin H to be used as first antibody (primary antibody) with the mark substance mark.In addition, with protein bound antibody, can use Protein G or albumin A post to be detected or measure in the screening of the present invention.

In screening method of the present invention, can use any test-compound, for example the product of cell extract, cell culture supernatant, organism of fermentation, marine organism extract, plant milk extract, purifying or rough albumen, peptide, non-peptide compound, synthetic micromolecular compound and natural compounds.In the context of the present invention, test-compound can also use multimedia any acquisition in the combinatorial library method known in the art, described method comprises (1) biology library, (2) parallel solid phase of space addressable or solution phase library (spatially addressable parallel solid phase or solution phaselibraries), (3) need the synthetic library method of deconvolution (deconvolution), the synthetic library method that (4) " pearl one compound (one bead one compound) " library method and (5) use affinity chromatography to select.Wherein, the biology library method of using affinity chromatography to select only limits to peptide library, and other four kinds of methods are applicable to peptide, non-peptide oligomer or compound small molecules library (Lam (1997) Anticancer Drug Des.12:145-67).The present technique field that is found in for example of the method in synthetic molecules library (De Witt et al. (1993) Proc.Natl.Acad.Sci.USA 90:6909-13; Erb et al. (1994) Proc.Natl.Acad.Sci.USA 91:11422-6; Zuckermann et al. (1994) J.Med.Chem.37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew.Chem.Int.Ed.Engl.33:2059; Carell et al. (1 994) Angew.Chem.Int.Ed.Engl.33:2061; Gallop et al. (1994) J.Med.Chem.37:1233-51).Library of compounds can exist in solution (referring to Houghten (1992) Bio/Techniques 13:412-21), with (Lam (1991) Nature 354:82-4) on the pearl, chip (Fodor (1993) Nature 364:555-6), bacterium (United States Patent (USP) 5,223,409), spore (United States Patent (USP) 5,571,698; 5,403,484 and 5,223,409), plasmid (Cull etc. (1992) Proc.Natl.Acad.Sci.USA 89:1865-9) or phage (Scott and Smith (1990) Science 249:386-90; Delvin (1990) Science 249:404-6; Cwirla etc. (1990) Proc.Natl.Acad.Sci.USA 87:6378-82; Felici (1991) J.Mol.Biol.222:301-10; U.S. Patent application 2002103360).

Can be used for suppressing or strengthen activity by screening method isolated compound of the present invention, treat or prevent RCC etc. for example to result from the disease of cell proliferation disorders by the marker gene encoded polypeptides.In addition, by the compound that screening method of the present invention obtains, also comprise part-structure in the compound that screening method of the present invention obtains add, lack and/or replace and must compound.

Be used for the treatment of or prevent the pharmaceutical composition of RCC

The invention provides the composition that is used for the treatment of or prevents renal cell carcinoma, it contains any compound of selecting by the screening method of the invention described above.

When will be by screening method isolated compound of the present invention as medicament administration in people or other Mammals, for example when mouse, rat, cavy, rabbit, cat, dog, sheep, pig, ox, monkey, baboon, chimpanzee, isolated compound can directly be used, and perhaps can utilize the known drug preparation method to be mixed with formulation.For example, as required, it is oral that described medicine can be used as sugar coated tablet, capsule, elixir and microcapsule; Perhaps water or the acceptable liquid dosage of any other pharmacy become sterile solution or suspension, with the non-orally ingestible of the form of injection.For example, can be in the required unit dosage of generally accepted medicament administration mode (unit dose) with compound, mix with pharmaceutically acceptable carrier or medium, described carrier or medium specifically have sterilized water, physiological saline, vegetables oil, emulsifying agent, suspension agent, tensio-active agent, stablizer, seasonings, vehicle (excipient), vehicle (vehicle), sanitas, tackiness agent etc.According to the amount of activeconstituents in these preparations, can obtain the suitable dosage in stated limit.

The example that can be mixed into the additive in tablet and the capsule includes but not limited to tackiness agent such as gelatin, W-Gum, tragacanth gum (tragacanth gum) and gum arabic, vehicle is crystalline cellulose for example, swelling agent is W-Gum, gelatin and Lalgine (alginic acid) for example, lubricant is Magnesium Stearate for example, sweetener is for example peppermint (peppermint), Gaultheriaadenothrix oil and cherry of sucrose, lactose or asccharin and seasonings for example.When unit dosage is capsule, can also comprise liquid vehicle in the mentioned component, for example oil.The Injectable sterile composition can use for example distilled water for injection of solvent (vehicle), prepares according to the medicine manner of formulation of standard.

Physiological saline, glucose and comprise auxiliary material (adjuvants), other isotonic liquid as D-sorbyl alcohol, D-seminose, D-N.F,USP MANNITOL and sodium-chlor can be used as aqueous solution for injection.These can be used in combination with suitable solubilizing agent, and described solubilizing agent is such as alcohol, ethanol for example, and polyvalent alcohol is propylene glycol and polyoxyethylene glycol for example, and nonionogenic tenside is Polysorbate 80 (TM) and HCO-50 for example.

Sesame oil or soybean oil are the examples of oleaginous fluid, described oleaginous fluid can be used in combination as solubilizing agent with peruscabin or phenylcarbinol, also can be formulated together with damping fluid such as phosphate buffered saline buffer and sodium acetate buffer, analgesic agent example hydrochloric acid PROCAINE HCL, PHARMA GRADE, stablizer such as phenylcarbinol and phenol and/or antioxidant.The injection of preparation can be encased in the suitable ampoule (ampoule).

Can utilize method known in those skilled in the art to use pharmaceutical composition of the present invention to the patient, for example as intra-arterial, intravenously or percutaneous injection, or as in the nose, intramuscular or oral administration be applied to the patient.Application dosage and method are different because of patient's body weight and age and application process; But, those skilled in the art can select suitable application process routinely.If described compound can then can be inserted into this DNA gene therapy with in the carrier by dna encoding, and uses this carrier to treat to the patient.Dosage of using and method are different because of patient's body weight, age and symptom, but those skilled in the art can select them suitably.

For instance, when to the Orally administered and of the present invention protein binding of normal adult human (body weight 60kg) and when regulating its active compound, though have some differences according to its symptom, but dosage is generally the about 100mg of about 0.1mg-every day, be preferably the about 50mg of about 1.0mg-every day, the about 20mg of more preferably about 1.0mg-every day.

When with the injection form during to normal adult human (body weight 60kg) parenteral administration, though have some differences according to patient, target organ, symptom and medication, but way is the about 30mg of the about 0.01mg-of intravenous injection every day easily, the preferred about 20mg of about 0.1mg-every day, the more preferably from about dosage of the about 10mg of 0.1mg-every day.And under the situation of other animal, the conventional suitable amount of application of calculating can convert by the standard of 60kg body weight.

The experimenter's of renal cell carcinoma prognosis is suffered from assessment

The method of prognosis situation that the present invention also provides assessment to suffer from the experimenter of RCC, described method comprise the step of comparing with reference to identical RCC Expression of Related Genes in the cell colony with one or more RCC Expression of Related Genes in the population of test cells and the patient who is derived from a series of disease stages.By population of test cells relatively and genetic expression with reference to one or more RCC genes involveds in the cell colony, or reference source expression of gene in experimenter's population of test cells is graphic in time, can assess experimenter's prognosis situation.

For example, compare with normal control, the RCC genes involved of one or more downward modulations, for example the expression decreased of RCC252-972 perhaps, is compared with normal control, the RCC genes involved of one or more rises, for example the expression of RCC 1-251 increases, and the expression prognosis is not good.On the contrary, the expression of one or more RCC genes involveds (for example RCC 1-972) is similar to normal control, and expression experimenter prognosis is better.Preferably, can assess experimenter's prognosis situation by the expression pattern that compares RCC 1-972.

Test kit

The present invention also comprises the RCC detection reagent, for example specificity in conjunction with or identify one or more RCC nucleic acid nucleic acid (such as with a part of complementary oligonucleotide sequence of RCC nucleic acid) or can with the protein bound antibody by the RCC nucleic acid encoding.Described detection reagent can be with the packaged of test kit together.For example, detection reagent can be packaged in respectively in the different containers, for example nucleic acid or antibody (can combine, or with it is separated packing with matrix bonded reagent), contrast agents (positive and/or negative) and/or detectable mark with solid substrate.Can also comprise the explanation (as printed instructions, tape, VCR, CD-ROM etc.) of implementing described mensuration in the test kit.The mensuration pattern of test kit can be Northern hybridization known in the art or sandwich ELISA.

For example, the RCC detection reagent can be fixed on solid substrate such as the porous slip (porous strip), to form at least one RCC detection site.The mensuration district or the detection zone of porous slip can comprise a plurality of sites, and nucleic acid is all contained in each site.Test strip also can contain the site of feminine gender and/or positive control.Perhaps, control site can be arranged on the slip different with test strip.Randomly, different detection site can contain the immobilized nucleic acids of different amounts, and promptly first detection site amount is higher, and site amount subsequently is less.After adding given the test agent, demonstration can be surveyed the quantitative target (quantitative indication) of the amount of the RCC that exists in the number of loci sampling of signal.The shape of detection site can be any suitable detectable shape, is generally the strip or the point-like of crossing over the test strip width.

Perhaps, test kit also comprises nucleic acid matrix (nucleic acidsubstrate) array that contains one or more nucleic acid.One or more nucleotide sequences of being represented by RCC 1-972 are identified on nucleic acid specificity ground on the array.By identifying 2,3,4,5,6,7,8,9,10,15,20,25,40,50 or the more kinds of expression of nucleic acids of representing by RCC 1-972 with the level that combines of array test slip or chip.The matrix array for example may reside on the solid substrate, " chip " described in described solid substrate such as the United States Patent (USP) 5,744,305 (its full content is incorporated this paper into as a reference).

Array and nucleic acid group (pluralities):

The present invention also comprises the nucleic acid matrix array of one or more nucleic acid.One or more nucleotide sequences shown in the corresponding RCC 1-972 in nucleic acid specificity ground on the array.By detecting and array bonded nucleic acid, can identify 2,3,4,5 shown in the RCC 1-972,6,7,8,9,10,15,20,25,40,50 or more kinds of expression of nucleic acids level.

The present invention also comprises group's (being the mixture of two or more nucleic acid) of isolating (isolated) multiple nucleic acid.Nucleic acid may reside in liquid phase or the solid phase, for example is fixed on the solid support as nitrocellulose filter.This group comprises the nucleic acid shown in one or more RCC 1-972.In a plurality of embodiments, the nucleic acid group comprises 2,3,4,5 shown in the RCC 1-972, and 6,7,8,9,10,15,20,25,40,50 or more kinds of nucleic acid.

The method that suppresses renal cell carcinoma

The present invention also provides the method for the treatment of or alleviate the RCC symptom among the experimenter by one or more expression of gene (or activity of its gene product) among one or more expression of gene (or activity of its gene product) among the minimizing RCC 1-251 or the increase RCC 252-972.Can be with suitable treatment compound preventative or therapeutic ground be applied to the experimenter who suffers from RCC or be in (or to RCC susceptible) in the RCC initiation potential.The clinical method of employing standard or by detecting one or more the unconventionality expression level (or activity of corresponding gene product) among the RCC 1-972 can identify above-mentioned experimenter.The example of suitable therapeutical agent includes but not limited to the inhibitor of cell cycle regulating, cell proliferation and protein kinase activity.

Methods of treatment of the present invention comprises the steps: to increase the expression and/or the activity of the gene product of one or more following genes: the normal cell of the same types of organization that is originated with the RCC cell is compared, and described expression of gene reduces (" downward modulation " or " crossing low the expression " gene) in the RCC cell.In these methods, use the compound of significant quantity that the experimenter is treated, one or more cross the amount of low expressing gene among the described compound raising experimenter.Administration can be whole body administration or topical.Suitable treatment compound included but not limited to the polypeptide product of low expressing gene, its bioactive fragment and the nucleic acid of encode and hanging down expressing gene and have the expression regulation element that permission expresses in the RCC cell; For example, improve the material of endogenic this type of expression of gene level of RCC cell (promptly raising the expression that one or more cross low expressing gene).Use such compound and can resist the influence of the low expressing gene of one or more unusual mistakes in experimenter's nephrocyte, and improve experimenter's clinical condition.

Perhaps, methods of treatment of the present invention can comprise the steps: to reduce the expression and/or the function of the gene product of one or more genes that abnormal expression increases in the RCC cell (" rise " or " the mistake high expression level " gene).Can express according to any inhibition the in the several method known in the art.For example, can suppress to express by use inhibition or one or more nucleic acid of crossing the expression of cance high-expression gene of antagonism to the experimenter, described nucleic acid for example destroys the antisense oligonucleotide or the siRNA of one or more expression of crossing cance high-expression gene.

Antisense polynucleotides:

As mentioned above, use antisense nucleic acid, can reduce the expression level of RCC 1-251 corresponding to the nucleotide sequence of RCC 1-251.Is useful corresponding to the antisense nucleic acid of the RCC 1-251 that raises in renal cell carcinoma for the treatment of renal cell carcinoma.Specifically, antisense nucleic acid of the present invention can by with RCC 1-251 or corresponding with it mRNA in conjunction with playing a role, thereby suppress described gene transcription or translation, promote described mRNA degraded and/or suppress the coded proteic expression of RCC 1-251, the function of arrestin thus.The term that uses in this specification sheets " antisense nucleic acid " comprises and the complete complementary Nucleotide of target sequence and have the Nucleotide of one or more Nucleotide mispairing, if this antisense nucleic acid can with the target sequence specific hybrid.For example, antisense nucleic acid of the present invention comprises in those scopes at least 15 continuous nucleotides (span), have at least 70% or higher, preferred at least 80% or higher, more preferably at least 90% or higher, more preferably at least 95% or the polynucleotide of higher homology.Described homology can use algorithm known in the art to determine.

Antisense nucleic acid of the present invention acts on the proteic cell of generation by RCC correlating markings genes encoding in the following way: combine with this proteic DNA of coding or mRNA, suppressing it transcribes or translates, promote the expression of mRNA degraded and arrestin, thus the function of arrestin.

By mixing with suitable matrix, antisense nucleic acid of the present invention can be made external preparation, as liniment (liniment) or net for catching birds or fish agent (poultice), wherein said suitable matrix is inert to nucleic acid.

And, as required, by adding vehicle, isotonic agent (isotonic agents), solubilizing agent, stablizer, sanitas, analgesic agent etc., antisense nucleic acid of the present invention can be mixed with for example tablet, powder, particle, capsule, liposome methods, injection, solution, nasal drop and freeze-dried.These formulations can be by following known method preparation.

Use antisense nucleic acid of the present invention to the patient, can directly it be imposed on the affected part, also it can be imported blood vessel so that it arrives the affected part.Can also use antisense sealing medium (antisense-mountingmedium) can improve persistence and membrane permeability.Example includes but not limited to liposome, poly-L-Lysine, lipid, cholesterol, lipofection reagent (lipofectin) or their derivative.

The dosage of antisense nucleic acid of the present invention can suitably be adjusted according to patient's situation, and uses with required amount.For example, the dosage range that can use is 0.1 to 100mg/kg, and preferred 0.1 to 50mg/kg.

Antisense nucleic acid of the present invention suppresses the proteic expression of the present invention, thereby can be used to suppress the proteic biological activity of the present invention.In addition, the expression inhibitor that contains antisense nucleic acid of the present invention also can be used to suppress proteic biological activity of the present invention.

Antisense nucleic acid of the present invention comprises modified oligonucleotide.For example, use thion acidifying (thioated) oligonucleotide can make oligonucleotide have the nuclease resistance.

Can use the expression level that reduces marker gene at the siRNA of marker gene.In this specification sheets, term " siRNA " refers to stop the double stranded rna molecule of said target mrna translation.Employing is the standard technique of siRNA transfered cell, and this is comprising being the technology of the template of rna transcription with DNA.SiRNA comprise following one of both or both: adopted RCC 1-251 (for example C6700 (RCC81), B7032N (RCC126) or B9320 (RCC173)) nucleotide sequence is arranged, antisense RCC 1-251 (for example C6700, B7032N or B9320) nucleotide sequence.SiRNA can be made of two complementary molecules; Also can make up siRNA has from the adopted sequence of having of target gene and complementary antisense sequences, for example hair clip its single transcript; In some embodiments, it causes Microrna (microRNA, generation miRNA).

In target cell, siRNA with combine corresponding to the transcript one of among the RCC 1-251, the albumen that causes cell to produce reduces.The length of oligonucleotide is at least 10 Nucleotide, can be isometric with naturally occurring transcript.The length of preferred oligonucleotide is 75,50,25 Nucleotide of less than.More preferably the length of oligonucleotide is 19-25 Nucleotide.

Method of the present invention can be used for changing genetic expression at the cell of C6700, B7032N or B9320 rise (for example because due to the malignant transformation of cells).In target cell, siRNA combines with C6700, B7032N or B9320 transcript, causes C6700, the B7032N of cell or B9320 to produce minimizing.The length of oligonucleotide is at least about 10 Nucleotide, can be isometric with naturally occurring C6700, B7032N or B9320 transcript.In the present invention, the target sequence of siRNA can be selected from the nucleotide sequence of C6700V2.The major part of this nucleotide sequence is that two variants of C6700 are to have between V1 and the V2.Therefore, the siRNA that effectively suppresses a variant also may suppress another variant.Like this, the siRNA of inhibition C6700 V1 and C6700 V2 can be used for treatment for cancer or prevention.Perhaps, the specific siRNA of C6700 V2 also can be used for treatment for cancer or prevention.The example of the siRNA oligonucleotide of the C6700 that inhibition C6700 expresses in mammalian cell comprises the oligonucleotide that comprises target sequence, and described target sequence for example may suppress the SEQ ID NO:43 of C6700 V1 and C6700 V2,47 or 81 Nucleotide.In the present invention, the target sequence of siRNA can be selected from the nucleotide sequence of B7032N.For example, contain the Nucleotide conduct of SEQ ID NO:106 and the siRNA oligonucleotide of the target sequence of B7032N.In the present invention, the target sequence of siRNA can be selected from the nucleotide sequence of B9320 V2.The major part of this nucleotide sequence is total between B9320 V1 and two variants of V2.Therefore, the siRNA that effectively suppresses a kind of variant may also suppress another kind of variant.Like this, the siRNA of inhibition B9320 V1 and B9320 V2 can be used for treatment for cancer or prevention.Perhaps, the specific siRNA of B9320 V2 also can be used for treatment for cancer or prevention.The example of the siRNA oligonucleotide of the B9320 that inhibition B9320 expresses in mammalian cell comprises the oligonucleotide that contains target sequence, and wherein said target sequence for example can suppress the Nucleotide of the SEQID NO:110 of B9320 V2.

Method of design with double-stranded RNA of the ability of inhibition of gene expression in target cell is known.Referring to No. the 6506559th, United States Patent (USP) for example, it incorporates this specification sheets into as a reference in full.For example, can (http://www.ambion.com/techlib/misc/siRNA_finder.html) obtain the siRNA designing computer programs from the Ambion website.It is synthetic that this computer program is used for siRNA based on following rules selected nucleotide sequence.

Select the siRNA target site:

1. the AUG initiator codon from the target transcript begins to scan downstream, seeks AA dinucleotides sequence.Write down 19 contiguous Nucleotide of the appearance of each AA and 3 ' side thereof as potential siRNA target site.Tuschl etc. are at (1999) Genes Dev 13 (24): among the 3191-7, do not recommend at 5 ' and the zone (within 75 bases) of 3 ' non-translational region (UTRs) and contiguous initiator codon design siRNA because aforementioned region may comparatively be rich in the modulin binding site.Conjugated protein and/or the translation initiation complex of UTR can disturb and the combining of siRNA endonuclease enzyme complex.

2. described potential target site and suitable genome database (people, mouse, rat etc.) are compared, will get rid of outside considering with any target sequence of the remarkable homologous of other encoding sequence.BLAST (Altschul SF, et al., (1997) Nucleic Acids Res. are adopted in suggestion; 25:3389-402; (1990) J Mol Biol.; 215:403-10.), it is found in NCBI server: www.ncbi.nlm.nih.gov/BLAST/.

3. select qualified target sequence to be used to synthesize.Typically, select several target sequences along the length of gene to be evaluated.

The present invention also comprises the isolated nucleic acid molecule of the nucleotide sequence that comprises target sequence, for example SEQ IDNO:43,47,81,106 or 110 Nucleotide, and with the nucleic acid molecule of the nucleic acid array complementation of SEQ ID NO:43,47,81,106 or 110 Nucleotide.In this manual, term " isolating nucleic acid " is taken out from its intrinsic environment (for example, being natural existence as if it, its natural surroundings), thereby is by the nucleic acid molecule that changes in artificial (synthetically) mode from its native state.In the present invention, the example of isolating nucleic acid comprises DNA, RNA and their derivative.When isolating nucleic acid is the RNA or derivatives thereof, should in nucleotide sequence, base " t " be replaced with " u ".In this specification sheets, term " complementary " refers to form Watson-Crick or Hoogsteen base pairing between the nucleotide unit of nucleic acid molecule, and term " combination " refers to two kinds of nucleic acid or compound or associated nucleic acid or compound or its physics or chemical interaction between making up.The complementary nucleotide sequence is hybridized under optimum conditions, forms the stable duplex that does not contain or do not contain mispairing substantially.When being used for purpose of the present invention, the mispairing of two sequences is no more than 5 and promptly thinks complementary.

In the context of the present invention, the sense strand of isolating Nucleotide of the present invention and antisense strand can form double chain nucleotide or hairpin ring structure by hybridization.In preferred embodiments, the mispairing that occurs in average per 10 pairings of such duplex is no more than 1.In particularly preferred embodiments, the chain of duplex is complementary fully, and such two strands does not contain mispairing.Nucleic acid molecule is less than 4725 or 4494 Nucleotide (for C6700), or is less than 3503 Nucleotide (for B7032N), or is less than 2539 or 3427 Nucleotide (for B9320).For example, the length of nucleic acid molecule is less than about 500, about 200, about 75 Nucleotide.The present invention also comprises the carrier that contains the nucleic acid of describing in one or more these specification sheetss, and the cell that contains this carrier.For for the DNA of the siRNA of C6700, B7032N or B9320 or these siRNA that encode, isolating nucleic acid of the present invention is useful.These nucleic acid are used for siRNA or during the DNA of this siRNA that encodes, sense strand preferably is longer than about 19 Nucleotide, more preferably is longer than about 21 Nucleotide.

The present invention is partly based on following discovery: compare with non-carcinous nephrocyte, transcribe variant C6700V2 and cross high expression level in renal cell carcinoma (RCC).C6700 has two kinds of different variants of transcribing.The cDNA length of C6700V1 or V2 is respectively 4725 or 4494 Nucleotide.The nucleic acid of C6700V1 or V2 and peptide sequence are shown in SEQ ID NO:63 and 64 respectively, or SEQ ID NO:65 and 66.Sequence data can also obtain by following accession number.

C6700V1：AY358124

C6700V2：BC077726

Transfection SEQ ID NO:43,47 or 81 siRNA cause the growth-inhibiting of RCC clone.C6700 is positioned karyomit(e) 3p21.1, its denotion is: Sema territory, 7 thrombospondins repeat (type 1 and type 1 sample), membrane-spanning domain (TM) and short cell matter territory (brain signal albumen 5B) (designed Sema domain, seven thrombospondin repeats (type 1 and type 1 like), transmembrane domain (TM) and short cytoplamic domain (Semaphorin 5B)), SEMA5B.Brain signal protein family member, SEMA5B for example is with the axon guidance (axonal guidance) relevant (Adams, et al., (1996) Mech.Dev.57:33-45.) in the neurodevelopment process.RT-PCR shows with the Northern engram analysis: compare with kidney proximal tubule epithelial cell, C6700 raises (Fig. 2 b and 3, right drawing) in 3 (A704, OS-RC-2 and TUHR14TKB) of 8 RCC clones (ACHN, 769-P, RXF-631L, TUHR10TKB, Caki-1, Caki-2,786-O and A-498).C6700 have two of constituting by 23 exons different transcribe variant, (Fig. 8 is a) to correspond respectively to C6700V1 and C6700V2.In this manual, found that the V2 variant crosses high expression level (Fig. 8 b) specifically in the RCC cell.

The present invention is partly based on following discovery: compare with non-carcinous nephrocyte, B7032N crosses high expression level in renal cell carcinoma (RCC).Long 3503 Nucleotide of B7032N.The nucleic acid of B7032N and peptide sequence are shown in SEQ ID NO:112 and 113.Sequence data can also obtain by following accession number.

NM_004567

The siRNA of transfection SEQ ID NO:106 causes the growth-inhibiting of RCC clone.B7032N censures and is (designed) PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6 diphosphatase 4; 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4) gene, sxemiquantitative RT-PCR shows: its clinical RCC case (Fig. 2 a) and rise in the RCC clone (data do not provide), but its be expressed in the inspected normal vital organ and all can not detect.The Northern engram analysis that carries out is subsequently confirmed: the B7032N transcript of about 3.5kb raises in 2 of 11 bladder cancer cell lines, but does not all express (Fig. 6) in the normal organ except that testis and pancreas.

The present invention is partly based on following discovery: compare with non-carcinous nephrocyte, transcribe variant B9320V2 and cross high expression level in renal cell carcinoma (RCC).B9320 has two kinds of different variants of transcribing.The cDNA length of B9320V1 or V2 is respectively 2539 or 3427 Nucleotide.The nucleic acid of B9320V1 or V2 and peptide sequence are shown in SEQ ID NO:115 and 116 respectively, or SEQ ID NO:118 and 119.Sequence data can obtain by following accession number.

B9320V1：BC034014

B9320V2：NM_153350

The siRNA of transfection SEQ ID NO:110 causes the growth-inhibiting of RCC clone.B9320 censures and is (designed) FBXL16 (F box and rich leucine repeat sequence protein 16) gene, sxemiquantitative RT-PCR shows: its RCC clinical case (Fig. 2 a) and rise in the RCC clone (data do not provide), but its be expressed in inspected any normal vital organ and all can not detect.The Northern engram analysis that carries out is subsequently confirmed: two kinds of B9320 transcripts---be B9320V1 (2.4kb) and B9320V2 (4kb) substantially, in 2 of 4 bladder cancer cell lines, significantly raise.The B9320V1 transcript is expressed in kidney and Tiroidina, and the B9320V2 transcript is not all expressed (Fig. 7) in the normal organ except that brain, testis, spinal cord and pancreas.

The structure of siRNA composition

The present invention relates to come cell growth inhibiting, i.e. growth of cancer cells by the expression that suppresses C6700, B7032N or B9320.For example, can suppress the expression of C6700, B7032N or B9320 by the siRNA (siRNA) of selectively targeted C6700, B7032N or B9320 gene.The target of C6700, B7032N or B9320 comprises SEQ ID NO:43,47,81 for example, 106 or 110 Nucleotide.

In the nonmammalian cell, proved double-stranded RNA (dsRNA) to genetic expression performance brute force and specific reticent effect, this is called RNA and disturbs (RNAi) (Sharp PA. (1999) GenesDev.; 13:139-41.).The enzyme that dsRNA is contained RNA enzyme III motif is processed into the dsRNA of 20-23 Nucleotide, is called siRNA (siRNA).By the multicomponent nucleic acid enzymes mixture, described siRNA specificity is target (Hammond SM, et al., (2000) Nature. with complementary mRNA; 404:293-6; Hannon GJ. (2002) Nature.; 418:244-51.).Verified, constitute by 20 or 21 aggressiveness dsRNA, have 19 complementary nucleotides and 3 ' terminal incomplementarity thymidine or the dimeric siRNA of uridine, in mammalian cell, has the low effect of striking of gene specific, and the globality of inducible gene expression does not change (Elbashir SM, et al., (2001) Nature.; 411:494-8.).And, the plasmid that contains small nuclear rna (snRNA) U6 or polymerase III H1-RNA promotor, can produce so little raising property of RNA III type class rna plymerase iii (type III class of RNApolymerase III) effectively, therefore can suppress its said target mrna (Miyagishi M and TairaK. (2002) Nat Biotechnol. in composition ground; 20:497-500.; Brummelkamp TR, et al., (2002) Science.296:550-3.).

In the context of the present invention, can contact cell growth inhibiting with the composition of the siRNA that comprises C6700, B7032N or B9320 by making cell.Further cell is contacted with transfection agents.The transfection agents that is fit to is well known to a person skilled in the art.The inhibition of cell growth refers to compare with the cell that is not exposed to composition, the slower or viability decline of the rate of propagation of described cell.The cell growth adopts the method that well known to a person skilled in the art to measure, for example the MTT cell proliferating determining.

The siRNA of C6700, B7032N or B9320 can be at the single target of C6700, B7032N or B9320 gene order.Perhaps, siRNA can be at a plurality of targets of C6700, B7032N or B9320 gene order.For example, composition can comprise at C6700, the B7032N of 2,3,4,5 or the more a plurality of target sequence of C6700, B7032N or B9320 or the siRNA of B9320.The target sequence of C6700, B7032N or B9320 is meant the identical nucleotide sequence of a part with C6700, B7032N or B9320 gene.Target sequence can comprise 5 ' untranslated (UT) district, open reading frame (ORF) or the 3 ' non-translational region of people C6700, B7032N or B9320 gene.Perhaps, siRNA is upstream or downstream instrumentality (modulator) the complementary nucleotide sequence with C6700, B7032N or B9320 genetic expression.The example of upstream or downstream instrumentality comprise with C6700, B7032N or B9320 gene promoter bonded transcription factor, with C6700, B7032N or the interactional kinases of B9320 polypeptide or Phosphoric acid esterase, C6700, B7032N or B9320 promotor or enhanser.The siRNA of C6700, B7032N or B9320 and said target mrna hybridization, and by be generally strand mRNA transcript and combine, disturb protein translation, like this by disturbing proteic expression to reduce or suppressing generation by C6700, B7032N or the B9320 polypeptide of C6700, B7032N or B9320 genes encoding.Therefore, siRNA molecule of the present invention can define under stringent condition Yu from the mRNA of C6700, B7032N or B9320 gene or the ability of cDNA specific hybrid according to it.For the present invention, term " hybridization " or " specific hybrid " all are meant the ability that 2 nucleic acid molecule are hybridized down in " tight hybridization conditions ".Phrase " tight hybridization conditions " refers to following condition, and nucleic acid molecule under this condition usually in the complex mixture of nucleic acid, with its target sequence hybridization, but but the hybridization of detection level does not take place with other sequence.Stringent condition depends on sequence, and is different in varying environment.Longer sequence is specific hybrid under comparatively high temps.Comprehensive guide about nucleic acid hybridization sees Tijssen, (1993) Techniques in Biochemistry and MolecuklarBiology--Hybridization with Nucleic Probes, " Overview of principles ofhybridization and the strategy of nucleic acid assays ".Usually, the stringent condition of selection is the pyrolysis chain temperature (T than specified sequence under the ionic strength that limits, pH _m) low about 5-10 ℃.T _mBe have 50% with target complementary probe under the equilibrium state with the temperature of target sequence hybridization (under ionic strength, pH and the nucleic acid concentration that is limiting) (when target sequence is excessive, T _mUnder the temperature, there is 50% the probe occupied under equilibrium state).In addition, by adding destabilizing agent, also can reach stringent condition as methane amide.For selectivity or specific hybridization, positive signal at least 2 times to background, be preferably 10 times to background hybridization.Exemplary tight hybridization conditions can be as follows: in 50% methane amide, 5x SSC, 1%SDS in 42 ℃ of insulations, perhaps in 5x SSC, 1%SDS in 65 ℃ of insulations, wash in 50 ℃ with 0.2x SSC, 0.1%SDS then.

In the context of the present invention, the length of siRNA is preferably and is less than about 500, about 200, about 100, about 50 or about 25 Nucleotide.Preferably, the length of siRNA is about 25 Nucleotide of about 19-.The examples of nucleic acid that is used to prepare C6700, B7032N or B9320 siRNA comprises the sequence of SEQ ID NO:43 as target, 47,81,106 or 110 Nucleotide respectively.And, in order to strengthen the inhibition activity of siRNA, Nucleotide " u " can be added to 3 ' end of the antisense strand of target sequence.The number of " u " to be added is at least about 2, and it is about 10 to be generally about 2-, is preferably about 2-about 5." u " that adds forms strand at 3 ' end of the antisense strand of siRNA.

Cell is to express or cross the arbitrary cell of high expression level as the C6700 gene, B7032N or the B9320 that transcribe variant of C6700V2.Cell is epithelial cells such as nephrocyte.Perhaps, cell is a tumour cell, for example cancer, gland cancer, blastoma, leukemia, myelomatosis or sarcoma.Cell is a renal cell carcinoma.

The siRNA of C6700, B7032N or B9320, can with can with the direct transfered cell of mRNA transcript bonded form.Perhaps, the DNA of the siRNA of coding C6700, B7032N or B9320 can be included in the carrier.

For example, be cloned into by the form (by transcribing of dna molecular) that C6700, B7032N or B9320 target sequence all can be expressed with two chains and prepare above-mentioned carrier in the expression vector, described expression vector has and is positioned at C6700, B7032N or regulating and controlling sequence (Lee B9320 target sequence flank, that can be operatively connected, N.S., et al., (2002) Nature Biotechnology 20:500-5.).Transcribe by first promotor (for example being positioned at the promoter sequence of the DNA 3 ' side of being cloned) with the RNA molecule of C6700, B7032N or B9320 mRNA antisense, and the sense strand RNA molecule of C6700, B7032N or B9320 mRNA is transcribed by second promotor (for example being positioned at the promoter sequence of the DNA 5 ' side of being cloned).Described sense strand and antisense strand are hybridized in vivo, produce the siRNA construct that is used for reticent C6700, B7032N or B9320.Perhaps, can utilize two constructs to prepare the sense strand and the antisense strand of siRNA construct.The construct that clone's C6700, B7032N or B9320 codified have secondary structure (for example hair clip), wherein, single transcript has concurrently from adopted sequence of having of target gene and complementary antisense sequences.

In order to form hairpin ring structure, can between by adopted sequence and antisense sequences, place the ring sequence of forming by any nucleotide sequence (loop sequence).Therefore, the present invention also provides has general formula 5 '-siRNA of [A]-[B]-[A ']-3 ', and wherein, [A] is the ribonucleoside acid sequence, its corresponding to sequence from mRNA or the cDNA specific hybrid of C6700, B7032N or B9320.In preferred embodiment, [A] for the corresponding ribonucleoside acid sequence of sequence that is selected from SEQ ID NO:43,47,81,106 or 110 Nucleotide, [B] about 3～the ribonucleoside acid sequence that about 23 Nucleotide are formed of serving as reasons, the ribonucleoside acid sequence that [A '] is made up of the complementary sequence of [A].

Zone [A] and [A '] hybridization form the ring by zone [B] formation then.The length of described ring sequence is preferably about 3-23 Nucleotide.Described ring sequence for example can be selected from the group of being made up of following sequence (http://www.ambion.com/techlib/tb/tb_506.html).In addition, the ring sequence of being made up of 23 Nucleotide also provides active siRNA (Jacque, JM., et al., (2002) Nature 418:435-8.).

CCC, CCACC or CCACACC:Jacque, JM., et al., (2002) Nature 418:435-8.

UUCG：Lee，NS.，et al.，(2002)Nature Biotechnology 20：500-5.Fruscoloni，P.，et al.，(2003)Proc.Natl.Acad.Sci.USA 100：1639-44.

UUCAAGAGA：Dykxhoorn，D.M.，et al.，(2003)Nature ReviewsMolecular Cell Biology 4：457-67。

For example, it is as follows to have a preferred siRNA of hairpin ring structure of the present invention.In following structure, the ring sequence can be selected from CCC, UUCG, CCACC, CCACACC and UUCAAGAGA.Preferred ring structure is UUCAAGAGA (being " ttcaagaga " among the DNA).

CTACCTCTTCAGACTCAGC-[B]-GCTGAGTCTGAAGAGGTAG (for SEQ ID NO:43 target sequence)

CCATGTGAGCACTTGGATG-[B]-CATCCAAGTGCTCACATGG (for SEQ ID NO:47 target sequence)

GCAGCAACGTTGCAGCACA-[B]-TGTGCTGCAACGTTGCTGC (for SEQ ID NO:81 target sequence)

GAGGACAATCCAGACGGCT-[B]-AGCCGTCTGGATTGTCCTC (for SEQ ID NO:106 target sequence)

GGAGCTCTACAACGTGCTG-[B]-CAGCACGTTGTAGAGCTCC (for SEQ ID NO:110 target sequence)

The adjusting sequence of C6700, B7032N or B9320 sequence flank can be the same or different, but should be able to regulate and control these genetic expression independently or with timeliness or spatiality mode.By the template of C6700, B7032N or B9320 gene is cloned into respectively on the carrier, siRNA is able to transcribe in cell, and wherein said carrier for example contains rna plymerase iii transcriptional units or the people H1 RNA promotor from small nuclear rna (snRNA) U6.For with the carrier transfered cell, can use transfection toughener (transfection-enhancing agent).FuGENE (Rochediagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen) and Nucleofactor (Wako pure Chemical) can be used as the transfection toughener.

According to standard method, reduce in tumour cells the ability that (for example, using kidney cell lines such as renal cell carcinoma (RCC) clone) C6700, B7032N or B9320 produce at the oligonucleotide of a plurality of parts of vitro detection C6700, B7032N or B9320 mRNA or with these part complementary oligonucleotide.Cultured cells is compared when not having the candidate set compound, with the minimizing of C6700, B7032N or B9320 transcript product in the cell that described candidate siRNA composition contacts, can use C6700, B7032N or B9320 specific antibody or adopt other to detect strategy to detect.For external based on cell mensuration or cell-less measurement in reduce the sequence that C6700, B7032N or B9320 produce, further measure the restraining effect of its cell growth.For external based on cell mensuration or cell-less measurement in cytostatic sequence, in rat or mouse, carry out measuring in the body, to confirm the minimizing that C6700, B7032N or B9320 produce and to suffer from the minimizing of growth of tumour cell in the animal of malignant tumour.

The methods of treatment of malignant tumour

By using the siRNA of C6700, B7032N or B9320, can treat the too high patient who is expressed as the tumour of feature with C6700, B7032N or B9320.For example, the siRNA therapy can be used for suppressing suffering from or the expression of risky generation renal cell carcinoma (RCC) patient C6700, B7032N or B9320.Employing can be identified such patient at the standard method of specific tumors type.Adopt for example computerized tomography (CT), nuclear magnetic resonance (MRI), ERCP (ERCP), Magnetic Resonance Cholangiopancreatography (MRCP) or supersonic method, can diagnose renal cell carcinoma (RCC).During clinical benefit such as the size of up-regulated gene expression decreased or tumour, morbidity (prevalence) or metastatic potential reduction, it is effective in treatment brings subject.When using described treatment, " effectively " refers to treat the formation that postpones or prevent tumour when preventative, perhaps postpones, prevents or the clinical symptom of ameliorate tumor.Can combine to determine validity with the method for any known diagnosis or treatment specific tumors type.

The siRNA treatment can be undertaken by following mode: by the standard vector of the siRNA of the present invention that encodes, and/or for example synthesize sending of siRNA molecule by gene delivery system and pass, siRNA is applied to the patient.Typically, will synthesizing property siRNA molecular chemistry stabilization, to prevent that it is in vivo by nuclease degradation.The preparation method of chemical stabilization RNA molecule is well-known in the art.Typically, such RNA molecule comprises modified skeleton and Nucleotide, to prevent the rnase effect.And other modification also is possible, and for example cholesterol link coupled siRNA shows the pharmacological property (Song et al. (2003) Nature Med.9:347-51) that improves.Particularly this wherein, suitable gene delivery system comprises liposome, the receptor-mediated virus vector such as delivery system or simplexvirus, retrovirus, adenovirus, adeno associated virus that send.

Treatment can be formulated in the pharmaceutically acceptable carrier with nucleic acid composition.Therapeutic composition can also comprise such as above-mentioned gene delivery system.Pharmaceutically acceptable carrier is to be suitable for the biological fitness solvent used to animal, for example physiological saline.The treatment significant quantity of compound is to obtain the amount of ideal medical outcome in the animal of treatment, for example reduce C6700, B7032N or B9320 gene product generation, for example reduce cell growth or minimizing tumor growth such as propagation.

Parenteral administration for example intravenously, subcutaneous, intramuscular and intraperitoneal send the approach of passing, and can be used to send the siRNA that passs C6700, B7032N or B9320 composition.For the treatment of renal cell carcinoma, Renal artery direct injection is particularly preferred.

Dosage for arbitrary patient depends on multiple factor, comprises patient's stature (size), body surface area, age, concrete nucleic acid, sex, administration time and the route of administration used, general health situation and the other medicines of using simultaneously.The intravenously application dosage of nucleic acid is about 10 ⁶～10 ²²The nucleic acid molecule of individual copy.

Polynucleotide of the present invention can adopt standard method to use, for example, be expelled to tissues such as muscle or skin between in the matter space, import the recycle system or body cavity or by sucking or being blown into.In addition polynucleotide by injection, perhaps can send with the acceptable liquid vehicle of pharmacy of water-based or part water-based and pass to animal usually.Polynucleotide can make up with liposome (for example positively charged ion or anionic liposome).Polynucleotide of the present invention are included in and express necessary gene information, for example promotor in the target cell.

Antisense oligonucleotide of the present invention or siRNA suppress polypeptide expression of the present invention, thereby can suppress the biological activity of or several polypeptide of the present invention.In addition, the expression inhibitor that comprises antisense oligonucleotide of the present invention or siRNA can suppress the biological activity of polypeptide of the present invention, thereby is useful.Therefore, the composition that comprises antisense oligonucleotide of the present invention or siRNA can be used for the treatment of renal cell carcinoma.

Perhaps, combine with gene product or the compound of suppressor gene product function otherwise, can be suppressed at the function of one or more gene products of the gene of crossing high expression level among the RCC by using.For example, described compound can be a gene product bonded antibody of crossing high expression level with one or more.

The present invention relates to antibody particularly at by raising the coded proteic antibody of marker gene or the segmental purposes of described antibody.As using in this specification sheets, term " antibody " refers to have the immunoglobulin molecules of specificity structure, its only with the antigen (promptly raising the gene product of marker gene) that is used for synthetic this antibody or with its AI that is closely related (promptly combining).In addition, antibody can be the antibody of antibody fragment or modification, as long as it is in conjunction with one or more marker gene encoded protein.For example, described antibody fragment can be Fab, F (ab ') ₂, Fv or the strand Fv (scFv) (Huston et al., (1988) Proc NatlAcad Sci USA 85:5879-83) that will be formed by connecting by suitable joint from the Fv fragment of H chain and L chain.More specifically, can use enzyme such as papoid or pepsin antibody to produce antibody fragment.Perhaps, can also make up the gene of this antibody fragment of coding, be inserted into suitable expression vector, and in proper host cell, express (referring to for example Co MS.et al., (1994) J.Immunol.1 52:2968-76; Better M.and Horwitz AH. (1989) MethodsEnzymol.178:476-96; Pluckthun A.and Skerra A. (1989) Methods Enzymol.178:497-515; Lamoyi E. (1986) Methods Enzymol.121:652-63; Rousseaux J.etal., (1986) Methods Enzymol.121:663-9; Bird RE.and Walker BW. (1991) Trends Biotechnol.9:132-7.).

Antibody can be by modifying with various molecules such as polyoxyethylene glycol (PEG) coupling.The invention provides such modified antibodies.Can obtain modified antibodies by antibody is carried out chemically modified.These modifying method are this area routines.

Perhaps, can obtain the chimeric antibody that antibody conduct of the present invention has the variable region that is derived from the non-human antibody and is derived from the constant region of people's antibody, perhaps as containing the complementary determining region (CDR) that is derived from the non-human antibody, the framework region (FR) that is derived from people's antibody and the humanized antibody of constant region.Described antibody can utilize the known technology preparation.

Pointing to the cancer therapy that the specific molecular that takes place in the cancer cells changes is identified as effectively by the clinical development of following anticarcinogen and the approval of administration: the trastuzumab (trastuzumab) that is used for the treatment of advanced breast cancer (Herceptin), the imatinib mesylate (imatinib methylate) that is used for the treatment of chronic lymphocytic leukemia (Gleevec) is used for the treatment of the gefitinib (Iressa) of nonsmall-cell lung cancer (NSCLC) and is used for B cell lymphoma and the lymphadenomatous rituximab of amphicyte (mantle cell) (anti-CD20 mAb) (Ciardiello F and Tortora G. (2001) Clin Cancer Res.; 7 (10): 2958-70.Review.; Slamon DJ, et al., (2001) N Engl J Med.; 344 (11): 783-92.; Rehwald U, et al., (2003) Blood.; 101 (2): 420-4.; Fang G, et al., (2000) .Blood, 96,2246-53.).Be effectively on these clinical drugs, and have better tolerance, because their target cell transformed only than traditional carcinostatic agent.Therefore, these medicines have not only improved cancer patients's survival rate and quality of life, and have proved the reasonableness of molecular targeted cancer therapy theory.In addition, when targeted drug and standard chemotherapy are used in combination, can strengthen effectiveness (Gianni L. (2002) .Oncology, 63 Suppl 1, the 47-56. of standard chemotherapy; Klejman A, et al., (2002) .Oncogene, 21,5868-76.).Therefore, Wei Lai cancer therapy will relate to probably with conventional medicine with make up at the target thing specific reagent of tumour cell different qualities such as vasculogenesis (angiogenesis) and wetting property (invasiveness).

These control methods can exsomatize (ex vivo), in external (for example by culturing cell in the presence of medicine) or the body (for example by giving experimenter's drug administration) carry out.Described method comprises the therapy as the abnormal activity of the unconventionality expression of antagonism (counteract) difference expression gene or its gene product, the combination of using albumen or proteic combination or nucleic acid molecule or nucleic acid molecule.

Can treat disease and illness with one or more active therapeutical agents of crossing cance high-expression gene of antagonism (promptly reducing or inhibition), described disease and illness are characterised in that the expression level of gene and gene product or biological activity (with respect to the experimenter who does not suffer from disease or illness) increase respectively to some extent.Can therapeutic or prophylactically use the therapeutical agent of antagonistic activity.

Therefore, the therapeutical agent that can be used for the context of the invention for example comprises polypeptide or its analogue, derivative, fragment or the homologue of (i) RCC genes involved; The antibody that (ii) resisted cance high-expression gene or gene product; High expression level or the mistake of (iii) encoding hanged down the nucleic acid of one or more genes of expressing; The (iv) nucleic acid of antisense nucleic acid or " dysfunction (dysfunctional) " (that is, because the allos in one or more cross the nucleic acid of cance high-expression gene insert due to); (v) siRNA (siRNA); Or (vi) conditioning agent (that is, changed high expression level or cross low express polypeptide and combine interactional inhibitor, agonist and antagonist between the counterpart (binding partner) with it).The endogenous function (referring to for example Capecchi, (1989) Science 244:1288-92) that handicapped antisense molecule is used for coming by homologous recombination " knocking out " polypeptide.

With level or biological activity decline (with respect to the experimenter who does not suffer from disease or illness) is the disease and the illness of feature, can use the therapeutical agent that improves active (that is, to its active agonist) to treat.Can use in the mode of treatment or prevention and raise active therapeutical agent.Utilizable therapeutical agent includes but not limited to polypeptide (or its analogue, derivative, fragment or homologue) or improves the agonist of bioavailability (bioavailability).

By obtaining patient tissue samples (for example from biopsy), and in the structure and/or the activity of the peptide of its RNA of external test or peptide level, the expression mRNA of the gene that changes to some extent (or express), come quantitation of peptides and/or RNA, thereby can easily detect the rising or the decline of level.Method well known in the art includes but not limited to immunoassay (for example carrying out sodium laurylsulfonate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry etc. behind Western engram analysis, immunoprecipitation) and/or detects the hybridization analysis (for example Northern analysis, dot blotting, in situ hybridization etc.) that mRNA expresses.

Preventive administration carries out before the obvious clinical symptom of disease occurring, with the appearance that stops disease or illness or postpone its process.

Therapy of the present invention can comprise the step that cell is contacted with conditioning agent, and described conditioning agent can be regulated the activity of the gene product of one or more difference expression genes.The example of protein-active conditioning agent includes but not limited to nucleic acid, albumen, these proteic naturally occurring connection parts (naturally-occurringcognate ligands), peptide, peptide mimics and other small molecules.For example, suitable medicine can stimulate one or more othernesses to cross one or more proteic activity of low expressing gene.

The invention still further relates to the method for renal cell carcinoma among treatment or the prevention experimenter, this method comprises the steps: described experimenter is used vaccine, and wherein said vaccine comprises by the immunologic competence fragment of the polypeptide of the nucleic acid encoding that is selected from RCC 1-251 (being the RCC up-regulated gene), described polypeptide or coding said polypeptide or its segmental polynucleotide.Use described polypeptide inducing antitumor immunity in the experimenter.For inducing antitumor immunity, giving has the experimenter who needs to use polypeptide by the nucleic acid encoding that is selected from RCC 1-251, the immunologic competence fragment of described polypeptide or coding said polypeptide or its segmental polynucleotide.Polypeptide or its immunologic competence fragment can be as the vaccines at RCC.In some cases, albumen or its fragment can be to be incorporated into TXi Baoshouti (TCR) or by antigen presenting cell (APC), to use as the form of scavenger cell, dendritic cell (DC) or B presented by cells.Because DC has strong antigen and presents ability, be most preferred so in APC, use DC.

In the present invention, the vaccine at RCC is meant the material with following ability: inducing antitumor immunity when it is inoculated in animal.According to the present invention, be considered to HLA-A24 or HLA-A*0201 restricted epitope peptide by RCC 1-251 encoded polypeptides or its fragment, it can induce the powerful and special immune response at the RCC cell of expressing RCC1-251.Therefore, the present invention also comprises the method for using the polypeptid induction antineoplastic immune.Generally speaking, antineoplastic immune comprises such as following immunne response:

The cytotoxic lymphocyte of-inducing antitumor,

-induce identification tumour antibody and

-inducing antitumor production of cytokines.

Therefore, when inducing any described immune response behind certain albumen inoculation animal, determine that this albumen has antineoplastic immune and induces effect.By protein induced antineoplastic immune can by in vivo or the immunity system among the observation in vitro host detect at this proteic replying.

For example, the inductive method of detection cytotoxic T lymphocyte is known.Particularly, the foreign matter that enters live body is presented to T cell and B cell by the effect of antigen presenting cell (APC).Respond to antigenic T cell that APC presented owing to this antigenic stimulation is divided into cytotoxic T cell (or cytotoxic T lymphocyte in the antigen-specific mode; CTL), breed (this is called the T cell activation) then.Therefore, the CTL of some peptides induces and can assess with inducing of CTL of detection by this peptide is presented to the T cell via APC.In addition, APC has the CD4+T of activation cell, CD8+T cell, scavenger cell, the effect of eosinophilic granulocyte (eosinophil) and NK cell.Because CD4+T cell and CD8+T cell also are important in antineoplastic immune, can use the activation effect of these cells to estimate the antineoplastic immune inducing action of peptide as index.

Using dendritic cell (DC) is well-known as the method for APC evaluate CT L inducing action in the art.In APC, DC is the representative APC with the strongest CTL inducing action.In the method, at first make and tried polypeptide and contact, make this DC and T cells contacting then with DC.With after DC contacts,, represent that then this is tried polypeptide and has inducing cytotoxic T cell activity if detect the T cell that has at the cytotoxic effect of target cell.The anti-tumor activity of CTL can for example adopt ⁵¹The cracking of the tumour cell of Cr mark (lysis) detects as index.Perhaps, adopt ³The method that H-thymidine picked-up activity or LDH (lactose desaturase) release are estimated the tumour cell degree of injury as index also is well-known.

Except that DC, peripheral blood lymphocytes (PBMC) also can be used as APC.Reported by under the situation that has GM-CSF and IL-4, cultivating PBMC and come inducing of enhanced CT L.Similarly, shown by under the condition that has keyhole limpet hemocyanin (KLH) and IL-7, cultivating PBMC and can induce CTL.

Can think the polypeptide that tried that is defined as having the CTL induced activity by these methods, be the polypeptide with DC activation effect and CTL induced activity subsequently.Therefore, induce polypeptide to can be used as antineoplastic vaccine at the CTL of tumour cell.In addition, also can be used as antineoplastic vaccine by contact the APC that obtains to induce at the ability of the CTL of tumour with described polypeptide.In addition, presenting polypeptide antigen by APC obtains Cytotoxic CTL and also can be used as antineoplastic vaccine.Utilization is called the cellular immunization therapy by these tumor therapeuticing methods of the antineoplastic immune that APC and CTL produce.

In general, when using polypeptide to be used for the cellular immunization therapy, known by make up multiple have the polypeptide of different structure and it contacted with DC can increase CTL inductive efficient.Therefore, when stimulating DC with protein fragments, it is favourable using polytype segmental mixture.

Perhaps, polypeptide can be confirmed by observing at the inducing of antibody generation of tumour inducing of antineoplastic immune.For example, induce the antibody that produces anti-this polypeptide in the laboratory animal with polypeptide immune, during these antibody inhibiting tumor cell growths, described polypeptide is considered as having the ability of inducing antitumor immunity.

Use vaccine-induced antineoplastic immune of the present invention, and this antineoplastic immune induce for the treatment and the prevention RCC possibility is provided.Anticancer therapy or can comprise any following step: the inhibition of cancerous cells growth, the shrinking back (involution) and inhibition that cancer takes place of cancer to the prevention of pathogenesis of cancer.Treatment for cancer or prevention comprise also that the individual death rate of suffering from cancer and sickness rate reduce, the alleviation etc. of the detected symptom that the tumor markers level reduces, cancer occurs together in the blood.Described therapeutic and prophylactic effects are preferably significant on the statistics.For example, under observation significance level is 5% or lower, in described observation vaccine is compared at the therapeutic or the prophylactic effects of cell proliferation disorders and the contrast of not using vaccine.For example, can be with Student ' s t-check, Mann-Whitney U-check or ANOVA are used for statistical analysis.

Above-mentioned have immunocompetent albumen or the coding this proteic carrier can make up with adjuvant.Adjuvant be meant when with have immunocompetent albumen common (or in succession) and strengthen compound when using at this proteic immunne response.The example of adjuvant includes but not limited to Toxins,exo-, cholera (cholera toxin), Salmonellas toxin (salmonella toxin), alum (alum) etc.In addition, vaccine of the present invention can make up with pharmaceutically acceptable carrier aptly.The example of such carrier includes but not limited to sterilized water, physiological saline, phosphate buffered saline buffer, nutrient solution etc.In addition, vaccine can comprise stablizer as required, suspension agent, sanitas, tensio-active agent etc.But vaccine whole body or use partly.Vaccine administration can comprise single-dose, perhaps comprises repeatedly strengthening administration (booster administrations).

When using APC or CTL as vaccine of the present invention, can be for example by method treatment or the prophylaxis of tumours of exsomatizing.More specifically, collect the experimenter's who receives treatment or prevent PBMC, cell is contacted under isolated condition with polypeptide, induce APC or CTL, then this cell is applied to the experimenter.Also can under isolated condition, import among the PBMC and induce APC by carrier with coded polypeptide.External evoked APC or CTL can clone before using.Have high target cell by clone and cultivation and destroy active cell, can more effectively implement the cellular immunization treatment.In addition, not only can be used for carrying out the cellular immunization treatment, can also be used at carry out the cellular immunization treatment from the tumour of other individual similar type at the experimenter that cell is provided with isolating APC of this mode and CTL.

Further, provide the pharmaceutical composition of the polypeptide of the present invention that contains pharmacy effective dose, it is used for the treatment of or prevents to comprise the cell proliferation disorders of cancer.This pharmaceutical composition can be used to excite antineoplastic immune.

Be used to suppress the pharmaceutical composition of RCC or pernicious RCC

Among the present invention, the appropriate drug preparation comprises and is suitable for the preparation that oral, rectum, nose, part (comprising oral cavity or hypogloeeis), vagina or non-digestive tract (comprising intramuscular, subcutaneous and intravenously) are used, and is suitable for by sucking or be blown into the preparation that (insufflation) uses.Preferred intravenously is used.Preparation can be chosen wantonly and be packaged in the discrete dose unit (dosage unit).

Being suitable for Orally administered pharmaceutical preparation comprises: capsule, cachet (cachet) or tablet, they respectively contain a certain amount of activeconstituents.The preparation that is fit to also comprises powder, particle, solution, suspension or emulsion.Activeconstituents can be chosen wantonly with the form of bolus sugar agent (bolus electuary) or paste (paste) and use.The tablet and the capsule that are used for oral administration can contain conventional excipients such as wedding agent, weighting agent, lubricant, disintegrating agent or wetting agent.Tablet can prepare by compression or moulding, wherein optional one or more system component that contains.Compressed tablets can prepare by the following method: with activeconstituents with free-pouring form (as powder or particle) randomly with wedding agent, lubricant, inert diluent, lubricant, tensio-active agent and/or dispersant, and in suitable machine, compress.The moulding tablet can prepare by the following method: in suitable machine the mixture that utilizes the moistening powder compounds of inert liquid diluent is carried out moulding.Described tablet can coat (coated) according to methods known in the art.The liquid oral prepared product can be the form of water-based or butyrous suspension liquid, solution, emulsion, syrup or elixir for example, and perhaps also can be used as desciccate provides, and water or other suitable vehicle constitute before use.Described liquid prepared product can contain conventional additives such as suspension agent, emulsifying agent, non-aqueous solvent (can comprise edible oil) and/or sanitas.Tablet can randomly be prepared so that the wherein slowly-releasing or the controlled release of activeconstituents to be provided.Can comprise a slice medicine of taking in every month in the packing of tablet.

Be suitable for the preparation that non-digestive tract uses and comprise water-based and nonaqueous aseptic injectable solution, wherein randomly contain antioxidant, buffer reagent, fungistat (bacteriostat) and make (isotonic) solute that the blood etc. of preparation and target recipient is opened; And water-based and nonaqueous sterile suspensions, wherein contain suspension agent and/or thickening material.Described preparation can provide with unitary dose or multidose container, for example Mi Feng ampoule and bottle; Can also under freeze-dried (freeze dried) condition, preserve, so only need to add just before use sterile liquid carrier for example salt solution, water for injection.In addition, can provide described preparation to be used for continuous infusion (continuous infusion).Promptly joining promptly, the injection solution and the suspension of usefulness can be prepared by the type of aforesaid sterilized powder, particle and tablet.

The preparation that is suitable for rectal administration comprises suppository (suppository), wherein comprises standard vector such as theobroma oil or polyoxyethylene glycol.Be suitable for a mouthful interior topical, the preparation that for example contains clothes or sublingual administration comprises lozenge (lozenge) and pastille (pastille), wherein lozenge is at flavoured base, as comprising activeconstituents in sucrose and gum arabic (acacia) or the tragacanth gum (tragacanth), and pastille is in matrix, as comprising activeconstituents in gelatin and glycerine or sucrose and the gum arabic.During intranasal administration, but compound of the present invention can be used as the liquid spray dispersed powders, or with the form of nasal drop (drop).Nasal drop can be chosen wantonly in this matrix and contain one or more dispersion agents, solubilizing agent and/or suspension agent with water-based or the preparation of non-aqueous matrix.

During inhalation, can carry compound of the present invention easily by the convenient means of insufflator, spraying gun, pressurized package (pressurized pack) or other conveying aerosol spray.Pressurized package can contain suitable propellant, as Refrigerant 12, trichlorofluoromethane, and dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can determine dose unit by the valve that conveying and metering amount (metered amount) is provided.

Perhaps, by sucking or when being blown into administration, compound can be the form of dry powder composite, for example the powdered mixture of this compound and suitable powder matrix (as lactose or starch).Powder composition can provide with unit dosage, and these formulations are capsule, cartridge case (cartridge), gel (gelatin) or foaming bag (blister pack) for example, by sucker or insufflator, can use described powder by above-mentioned formulation.

Other preparation comprises the implantable device and the adhesive patches (adhesivepatches) that can discharge therapeutical agent.

When needing, can adopt the above-mentioned preparation that is suitable for slow-release.Can also contain other activeconstituents in the pharmaceutical composition, such as biocide, immunosuppressor and/or sanitas.

Be to be understood that except that the above-mentioned composition of specifically mentioning preparation of the present invention can contain present technique field other reagent for described preparation type routine, the preparation that for example is suitable for oral administration can contain seasonings.

Contain activeconstituents in the preferred unit dosage preparation as following significant quantity or its suitable part.

For aforesaid various situations, can be with the dosage range of the about 250mg/kg of about 0.1-every day oral or by injection applying said compositions, for example polypeptide and organic compound.Adult's dosage range is generally the about 17.5g/ of about 5mg-days, is preferably the about 10g/ of about 5mg-days, most preferably is the about 3g/ of about 100mg-days.Tablet or other unit dosage that provides with dispersal unit can contain aptly with such dosage or as a plurality of such dosage effectively to be measured, and for example contains the about 500mg of the 5mg-that has an appointment, and is generally the about 500mg of about 100mg-.

Used dosage will depend on multiple factor, comprise experimenter's age and sex, the definite illness and the severity thereof of treatment.In addition, route of administration also can be dependent on illness and severity thereof and changes.Yet in any case, those skilled in the art can both calculate suitable optimal dose routinely on the basis of considering above-mentioned factor.

Further described aspect of the present invention in the following examples, these embodiment are not intended to limit the scope of putting down in writing in claims of the present invention.The following examples will describe the evaluation and the sign of the gene of differential expression in the RCC cell.

Use is dissected hyaline cell RCC cell and the normal renal cortical cell of selecting purifying by laser capture microdissection and is advanced The full genome cDNA microarray analysis of row renal cell carcinoma gene expression pattern

Use the cDNA microarray analysis gene expression pattern of showing 27648 genes, identified the tumor-marker and the target that are used for interventional therapy.Renal cell carcinoma is to exist as the solid block that comprises the various kinds of cell component.Therefore, adopt laser microbeam micro-dissection (LMM) purifying cancer cells from 15 routine renal cell carcinomas.Investigate its gene expression pattern, compare with the pattern of normal purifying renal cortical cell.These cell colonys are thus by homogenization (cell that is higher than 95% purifying).As a result, identifying 251 genes generally raises in the renal cell carcinoma cell.In these up-regulated genes, comprise the cyclin-D1 (CCND1) (Hedberg Y., et al., (1999) Int.J.Cancer, 84:268-72,1999.) that in RCC, crosses high expression level that has reported.This gene is crossed high expression level in 9 examples of of the present invention 15 routine information RCC cases.Identify the generally downward modulation in the renal cell carcinoma cell of 721 genes.

The present invention has overcome many restrictions of previous method.Therefore, gene expression pattern of the present invention has constituted very cancer reference accurately.At first, because the various kinds of cell composition does not exist only in and is present in the healthy tissues in the cancerous tissue yet, it is very big that the microarray analysis of use clinical sample before had been proved to be difficulty.Particularly, renal cell carcinoma has the advantages that the form with the solid block that comprises the various kinds of cell composition exists.Therefore, use the overall and normal renal cortical cell of renal cell carcinoma to carry out the remarkably influenced that the gene expression pattern analysis meeting is subjected to being examined the ratio of the cell mixing in the tissue, these ratios may be covered the remarkable increase or the minimizing of renal cell carcinoma genes involved.Therefore, in the present invention, use the LMM system from the surgery sample purifying the very cancer cells and the normal epithelium cell of high purity (more than 95%).Because adopt LMM even can finish the micro-dissection of individual cells level, this technology is important for the accurate microarray analysis of renal cell carcinoma sample.

Carefully purifying cancer cells and normally renal cortical cell, isolation of RNA then, carry out the cDNA microarray analysis, the result has identified that the gene that 251 expression are generally raised (can obtain expression data and the gene of expression ratio (Cy5/Cy3 strength ratio) greater than 5.0 in surpassing 50% cases of cancer; For can calculating in the case of 33-50%, and expression ratio is also assessed greater than 5.0 gene in computable whole cases.

The pattern that obtains in this specification sheets and describe is compared with early stage pattern and is improved to some extent, this is because they are to analyze by the colony to highly purified cancerous cells (renal cell carcinoma), and with maximally related normal control, promptly the high purity colony of renal cortical cell compares and obtains.Previous method and pattern are subjected to the very a high proportion of influence that mixes cell, and these cells can reduce the accuracy and the confidence level of preceding mode.Pattern of the present invention is the accurately full genomic gene expression pattern of first extensive renal cell carcinoma.These data have been identified the molecular target that is used for the adjusting of renal cell carcinoma treatment associated treatment and have been used for the early stage accurately new tumor-marker of specificity of diagnosis of cancer or precancerous condition.

Except as otherwise noted, all technology used herein and scientific terminology all with the ordinary technical staff in the technical field of the invention conventional understand equivalent in meaning.Although implementing or check can be used when of the present invention and similar or the method and the material that are equal to described herein, the description below appropriate means and material such as this specification sheets.All publications that this paper mentions, patent application, patent and other reference are all incorporated this paper into as a reference in full.Under the situation that has conflict, be as the criterion with this specification sheets that comprises definition.In addition, material described herein, method and embodiment only are unrestricted the present invention for explanation.

Embodiment

Materials and methods

Will describe the present invention further in following embodiment, this embodiment does not limit scope of the present invention in claims.

Evaluation derives from for example RCC of the tissue of diseased tissue and gene that healthy tissues is identified differential expression or morbid state.Measure according to following method:

The patient, tissue sample and clone

From 15 patients of informed consent (male sex's 9 examples wherein, women's 6 examples; The hyaline cell renal cell carcinoma comprises 10 routine pT1,2 routine pT2 and 3 routine pT3; The patient's age scope is 36 years old to 75 years old, 61.3 years old mean age) obtained renal cell carcinoma, all patients have given informed consent (table 1) to this.From medical records, obtained clinical information, and according to the classification of histopathology hypotype each tumour has been diagnosed by the pathology worker.Judged each patient's clinical stage according to Japanese urology meeting renal cell carcinoma Clinical pathological study general rule.All samples are freezing at once and be kept at-80 ℃.Will be from renal cell carcinoma (Caki-1, Caki-2,786-O, A498, ACHN, 769-P, A704, RXF-631 L, OS-RC-2, TUHR10TKB and TUHR14TKB) clone that obtains of cell and kidney proximal tubule epithelial cell (RPTEC) be with adding 10% foetal calf serum, the suitable culture medium monolayer culture of 1% microbiotic/anti-mycotic agent solution, and maintain 37 ℃ and contain 5%CO ₂Air in.

Table 1 RCC patient's clinical pathologic characteristic

Sample number	Age	Sex	pT
					1	57	M	1
2	70	M	2
				3	68	M	1
4	73	F	1
				5	54	M	1
6	71	F	1
				7	69	F	3
8	47	F	1
				9	75	M	3
10	41	M	1
				11	54	F	2
12	74	F	3
				13	66	M	1
14	62	M	1
				15	36	M	1

Tissue sample and LMM

Sample is embedded in the TissueTek OCT medium (Sakura), is kept at-80 ℃ then until use.The refrigerated sample is cut into the thin slice of 8 μ m continuously and with h and E it is dyeed to determine analyzed area with cryostat (cryostat).In order to prevent the crossed contamination of cancer cells and non-cancerous cells, utilize EZ Cut LMM System (SL Microtest GmbH) to prepare this two colonies, preparation is carried out according to the manufacturer's rules through some improvement.In order farthest to reduce tissue sampling and to organize influence in the preservation process, the cancer sample is to use same program handled.For the quality of checking R NA, the total RNA that extracts from each residue tissue has been carried out sex change (degenerative) agarose gel electrophoresis, their quality has been proved conclusively in the existence of ribosome-RNA(rRNA) band.

RNA extracts the RNA amplification that reaches based on T7

From each cell colony of laser capture, total RNA is extracted in the RLT lysis buffer (QIAGEN) of 350 μ l.At room temperature use the RNA 30 minutes that the DNA enzyme I (QIAGEN) of 30 units handle to extract.70 ℃ handle 10 minutes inactivations after, utilize the suggestion purifying RNA of RNeasy Mini Kit (QIAGEN) according to manufacturer.The RNA that utilizes Ampliscribe T7 Transcription Kit (EpicentreTechnologies) that all DNA enzyme I were handled has carried out the amplification based on T7.For each sample, two-wheeled amplification has produced the cloning RNA (aRNA) of 41.5-163.4 μ g, and when the contriver to from the time from the normal renal cortical cell cloning RNA of 11 patients with renal cell carcinoma, obtained 477.0 μ g altogether.Get respectively 2.5 μ g sample aliquot, they are existed under the condition of Cy5-dCTP and Cy3-dCTP (Amersham Biosciences) carrying out reverse transcription respectively from cancerous cells and normal renal cortical cell.

The cDNA microarray

In order to obtain to be used for the cDNA of point sample on slide glass, the contriver is according to preceding method (KitaharaO, et al., (2001) Cancer Res; 61:3544-9) each gene has been carried out RT-PCR.With high-density Microarray Spotter Lucidea (GE Healthcare, Amersham Biosciences) with PCR product point sample (GE Healthcare, Amersham Biosciences, Buckinghamshire UK) on VII type slide glass; With 9,216 genes in duplicate point sample on single slide glass.Three groups of different slide glasss (totally 27,648 gene points) have been prepared altogether; Also comprise identical 52 housekeeping genes and two negative controls in every group.According to preceding method (Okabe H, et al., (2001) CancerRes; 61:2129-37) prepared the cDNA probe from aRNA.In hybrid experiment, get 7.5 μ g from each cancerous tissue or from the cloning RNA (aRNA) that contrasts, under the condition that has Cy5-dCTP and Cy3-dCTP (GE Healthcare, Amersham Biosciences), it is carried out reverse transcription respectively.According to the described method of forefathers (Okabe H, et al., (2001) Cancer Res; 61:2129-37) hybridize the detection of washing and signal.

Hybridization and data are obtained

Except all steps are utilized Automated Slide Processor (Amersham Biosciences) implements, hybridization and washing all according to preceding method carry out (Ono K., et al., (2000) Cancer Res, 60:5007-11).

Utilize ArrayVision software (Imaging Research, Inc., St.Catharines, Ontario Canada) has carried out quantitatively from the Cy3 of 27,648 points and Cy5 strength of signal by the background method of replacement and has analyzed.Subsequently, the Cy5 (tumour) of each object point and the fluorescence intensity of Cy3 (contrast) are adjusted, make the average Cy3/Cy5 ratio of 52 housekeeping genes on the array equal 1.Owing to the confidence level of the data that obtain from low signal intensity is lower, the contriver has determined to hold back (cut-off) value (Ono according to preceding method on each slide glass, K., et al., (2000) Cancer Res, 60:5007-11.), and the gene that the strength of signal that Cy3 and Cy5 dyestuff are produced all is lower than cutoff value is excluded in (Saito-Hisaminato, A., et al. outside the further analysis, (2002) DNA Res, 9:35-45.).For other gene, the contriver utilizes the raw data of each sample to calculate the ratio of Cy5/Cy3.

The evaluation of the gene that generally raises among the RCC or reduce

The relative expression of each gene is included into one of four classes than (Cy5/Cy3 strength ratio): (A) raise (expression ratio＞5.0); (B) downward modulation (expression ratio＜0.2); (C) no change (expression ratio is between 0.2 and 5.0); (D) there is not expression (perhaps having a spot of expression still to be lower than the level of holding back that detects).Use these classification, detected the general vicissitudinous one group of gene of expression ratio in sample.In order to detect the general candidate gene that significantly raises or reduce in each group, at first to 27, the graphic screening of overall expression of 648 genes, select expression ratio＞5.0 or＜0.2 gene, and these genes be present in＞50% classified group in.

Sxemiquantitative RT-PCR

Picked out 19 up-regulated genes, and by sxemiquantitative RT-PCR The effects its expression level.(Life Technologies, Inc.) the 1-μ g aRNA sample aliquot reverse transcription that will derive from each sample becomes strand cDNA to utilize random primer and Superscript II.Dilute each cDNA mixture, be used for the primer shown in the use table 2 and carry out subsequent P CR amplified reaction.For with each expression of gene amount normalization method, from 52 housekeeping genes, selected farnesyl bisphosphate farnesyl transferase 1 (FDFT1) as internal contrast, because existing microarray data shows that the Cy5/Cy3 fluctuation of this gene is minimum.Amplification cycles number to the PCR reaction is optimized, and has guaranteed that product intensity is in the linear stage of amplified reaction.

Table 2. is used for the primer sequence of sxemiquantitative RT-PCR

LMMID	Accession number	Forward primer	SEQ ID NO	Reverse primer	SEQ ID NO
						A6149N	NM_018092	5′-GCTCTTATCAAGAATA ACAACTTCCA-3′	1	5′-AGTTATAGCGTCCAGG TCCAAC-3′	2
B0732	AA632745	5′-CAGAGTGCTTCCTCCA TCTCTTA-3′	5	5′-CATAAGAAGCATCGCT GAGAATC-3′	6
						B8940	NM_007250	5′-GAATCAAACTCAAGGC TGTGAAC-3′	7	5′-GAATGTGCCTCAAACT CAGAAAC-3′	8
C6314	W86513	5′-TCACCTGGTTTCAGTA CTGCTCT-3′	9	5′-GAAGGCACAGGTCTTG TAAAATG-3′	10
						C6700	BC077726	5′-TGACCTGTGCTTAGAA GTCCTTT-3′	11	5′-GTGTGTGTTCCTGAAC AGATGAA-3′	12
C7670	AA156409	5′-AAACAACATCTGACTT ACGGGAG-3′	13	5′-CAACATGAAGATTCTG AAGGGTC-3′	14
						C8379	W57613	5′-CCTCTACTTCCCATTCC ATTTCT-3′	15	5′-TGCACTACATATGGTC AGAGTGG-3′	16
C8919	CR749811	5′-GTTGCAGTTAGACGAA GTGGTTC-3′	17	5′-CCAAAAAGTTGTGCTA GTGTGTG-3′	18
						C9099	AW972553	5′-ATGATCTGCCCACTCA CCTC-3′	19	5′-AGGAGGCTAAAGGCA ATGAATAG-3′	20
D9553	AL832896	5′-GTGAATGAATGAGTGT GTCATGG-3′	21	5′-GTGTTAGTGTCACTCT CAGCCAG-3′	22
						F0887	AK021778	5′-GCCCTCAGAAGAGAA ACCTGTA-3′	23	5′-TCACCACTATGGTCGA TCTTTCT-3′	24
F4886	AK026403	5′-GCAGTCAGAAGGCAG GTTTC-3′	25	5′-TTCTTCCCACAACATCT CTATGC-3′	26
						F5749	AK025204	5′-GCAAATTCGGTAAAAC CCATC-3′	27	5′-CGAGACAGAAATAGG TTGCTGG-3′	28
A0707	NM_000677	5′-CCTGAAGGGTGCCTAG TTGA-3′	67	5′-ACTCAAAAACATCCAC AGGTGA-3′	68
						A4587	AF070609	5′-GGCTGTGGTGCAGTAA CCAT-3′	69	5′-CATCTACAAAGTAATG CTTCCCAGT-3′	70
B4578	AI290343	5′-TCCCTCACGTTATTGG AAGC-3′	71	5′-TCCACATCCTTCCTCA AAGG-3′	72

D9765	AA442590		5′-ATGATGGCCATTTTGA TGCT-3′	73	5′-GCTCTCCACGTTGGTA GGTC-3′	74
							A3412	NM_000552	5′-CACCAATGGCTCTGTT GTGT-3′	75	5′-TAAGAGCTCAGCCTTT ATTGTGG-3′	76
C4971	BC000234	5′-GCTACTACATGATTGG TGAGCAG-3′	77	5′-CTTTAATTGAGGTCAC AGGCATC-3′	78
						B7032N	NM_004567		5′-CTTCCAGACTCATCTG TCAGAGC-3′	117	5′-GCAACTCCTAACTCCC CTCTGTA-3′	87
B9320	BC000234	5′-CAGAGAGCCAGAGAG TGAGAGAG-3′	88	5′-GCATATACAGGAGAAT GAGGTCG-3′	89
						FDFT1	NM_004462	5′-AGTGAAATGCAGGTGA GAAGAAC-3′	29	5′-TCATTCTAGCCAGGAT CATACTAAG-3′	30

Because C6700 has 2 different transcripts, confirm which variant mistake high expression level in the RCC cell so use following primer to carry out sxemiquantitative RT-PCR:

C6700FV15’-GTCCCCACTGCTTTGAGATG-3’(SEQ ID NO：37)，

C6700RV15’-GTGTGTGTTCCTGAACAGATGAA-3’(SEQ ID NO：12)；

C6700FV25’-GACTGTGCAGGGTTCAATCTC-3’(SEQ ID NO：38)，

C6700RV25’-GTGTGTGTTCCTGAACAGATGAA-3’(SEQ ID NO：12)；

B2M (β 2MG) as internal contrast

5’-TTAGCTGTGCTCGCGCTACT-3’(SEQ ID NO：39)，

5’-TCACATGGTTCACACGGCAG-3’(SEQ ID NO：40)。

In order to investigate the expression level of middle C6700V1 of RCC cell (786-O, A704, OS-RC-2 and TUHR14TKB) and C6700V2, use following primer sets to carry out the RT-PCR experiment:

5 '-CTGGAAACAGCAGCCAGAG-3 ' (SEQ ID NO:83) and

5’-CAGTGCTGGCAAGACAGGTA-3’(SEQ ID NO：84)。Cycle number to the PCR reaction is optimized, and is in the logarithm of amplification in the stage to guarantee product intensity.

Use following primer sets further to confirm the expression level of the transcript of B9320 variant by RT-PCR: variant 1:5 '-GGAGTGGGAACCGCAGAAC-3 ' (SEQ ID NO:90), with 5 '-GACCCCCACCTTCAAATCAC-3 ' (SEQ ID NO:91), variant 2:5 '-GCTCGTGCTCGACAGGTGTGTA-3 ' (SEQ ID NO:92) and 5 '-CAGGTCATGGCCGGGTTC-3 ' (SEQ ID NO:93).Cycle number to the PCR reaction is optimized, and is in the logarithm of amplification in the stage to guarantee product intensity.

The Northern engram analysis

On 1% sex change sepharose, separate 1 μ g sample aliquot of following sample and transfer on the nylon membrane: use the various mRNAs of Micro-FastTrack (Invitrogen) from 4 renal cell carcinoma clones and RPTEC cellular segregation, and people's healthy tissues polyA (+) RNA (Clontech, Palo Alto, California), the heart, liver, lung, kidney, brain and testis.Make this Northern trace and people organize the Northern trace again (Clontech, Palo Alto is CA) with [α of the candidate gene that obtains by RT-PCR more ³²P]-hybridization of dCTP mark amplified production.Prehybridization, hybridization and cleaning are undertaken by supplier's suggestion.Use intensifying screen-80 ℃ of radioautograph 14 days trace.Specific probe is to use the Auele Specific Primer group shown in the table 3 to prepare by PCR.

Table 3. is used for the sequence of the primer of Northbern probe

LMMID	Accession number	Forward primer	SEQ ID NO	Reverse primer	SEQ ID NO
						C6700	BC077726	5′-CTGTGGACCTACT GGGCATT-3′	31	5′-GTGTGTGTTCCTGAA CAGATGAA-3′	12
F5749V1	AK025204	5′-TTTGCCTTTTTGTG TTTGCTT-3′	32	5′-CCAGAAGCCTGCAG TATACCA-3′	33
						F5749V2		5′-CTGGGATATGGCA GCAATGTA-3′	34	5′-ATATGCTGGCACTGT GTGGTC-3′	35
B7032N	NM_004567		5′-GGGTCAGAAAGTG TCTGGACTT-3′	94	5′-GCAACTCCTAACTCC CCTCTGTA-3′							87
						B9320	BC034104 NM_153350	5′-CAACAGCCCAAAG ATTTTCC-3′	95	5′-GGTCACGTGATAAA ATAGCACAA-3′	96
C8919	CR749811		5′-TCCAACCAAGTGG ACACTGA-3′	36	5′-CCAAAAAGTTGTGCT AGTGTGTG-3′							18

The structure of expression vector

With KOD-Plus archaeal dna polymerase (Toyobo, Osaka, Japan) obtained the open reading-frame (ORF) sequence of B7032N and B9320 by PCR, wherein used following primer: the B7032N-forward, 5 '-AACGAATTCATGGCGTCCCCACGGGA-3 ' (SEQ ID NO:97) (underscore is represented the EcoRI restriction enzyme sites) and 5 '-CAACTCGAGCTGGTGAGCAGGCACCGTG-3 ' (SEQ ID NO:98) (underscore is represented the XhoI restriction enzyme sites), with the B9320-forward, 5 '-CAAGAATTCATGTCGAGCCCGGGCATC-3 ' (SEQ ID NO, 99) and 5 '-GCTGAATTCACCTCAATGACGAGGCAGCGG-3 ' (SEQ ID NO, 100) (underscore is represented the EcoRI restriction enzyme sites).The PCR product of B7032N is inserted among the EcoRI and XhoI site of pCAGGSsnH3F expression vector.The PCR product of B9320 is inserted in the EcoRI site of pCAGGSsnH3F expression vector.Confirmed the dna sequence dna of these constructs by dna sequencing.

Immunocytochemical assay

With 5 * 10 ⁴The COS7 cell has been inoculated to carry out the exogenous expression in individual/hole.After 24 hours and 48 hours, use FuGENE 6 transfection reagents (Roche) according to manufacturer specification respectively with 1mgpCAGGS-HA-B7032N or pCAGGS-HA-B9320 transient transfection COS7 cell.Then, with the PBS (-) that contains 4% Paraformaldehyde 96 in 4 ℃ of fixed cells 15 minutes, and with 4 ℃ of processing of the PBS that contains 0.1%TritonX-100 2.5 minutes so that it is permeable.Subsequently, in 4 ℃ cell is covered 12 hours with the sealing non-specific hybridization with the PBS (-) that contains 3%BSA.Then, with the COS7 cell of the COS7 cell of B7032N-HA transfection and B9320-HA transfection rat anti HA antibody (SANTA CRUZ) incubation with dilution in 1: 1000.After cleaning with PBS (-), transfectional cell is dyeed with the AlexaFluro 488-coupling Chinese People's Anti-Japanese Military and Political College mouse second antibody (Molecular Probe) of diluting at 1: 3000.With 4 ', 6 '-diamidino-2-phenylindone two hydrochloric acid (4 ', 6 '-(DAPI) pair cell nuclear counterstaining of diamidine-2 '-phenylindolendihydrochrolide).(Japan) microscopically obtains fluoroscopic image for Leica, Tokyo at TCS SP2 AOBS.

The Western engram analysis

Use pCAGGS-nHA expression vector rotaring redyeing COS 7 cell as described above, make its exogenous expression's B7032N albumen and B9320 albumen respectively.Reclaim full cell lysate after 24 hours and 48 hours in transfection respectively.With transfectional cell cracking in lysis buffer (50mM Tris (pH 7.5), 150mmol/LNaCL, 10mmol/L CHAPS and 0.1% proteinase inhibitor mixture group III (Calbiochem)).After the homogenate, cell lysate is incubated 30 minutes on ice, centrifugal 15 minutes of 14000rpm, only making, supernatant separates with cell debris.(Bio-Rad, Hercules CA) estimate total protein concentration, then albumen are mixed with the SDS-sample-loading buffer, boil, and are loaded into the 10%SDS-PAGE gel then with protein determination kit.Behind the electrophoresis, Western blot is arrived on the nitrocellulose filter (GE Healthcare).To contain proteic film and seal, then with rat anti HA monoclonal antibody (SANTACRUZ) incubation, to detect external source B7032N albumen or B9320 albumen with confining liquid.Make protein band visual at last with film and the second largest murine antibody incubation of HRP link coupled, and by ECL detection reagent (GE Healthcare).

Set up the NIH3T3 cell of stably express PFKFB4

As mentioned above, with FUGENE6 B7032N expression vector, B9320 expression vector or analog carrier transfection are entered the NIH3T3 cell.With cells transfected incubation in containing 0.9mg/ml Geneticin (G418) substratum (Invitrogen).To clone the NIH3T3 cell and carry out subclone by limiting dilution.The expression of the B9320 of the B7032N of band HA label or band HA label is assessed by the Western engram analysis with anti-HA monoclonal antibody.At last, set up several clones, and called after B7032N-NIH3T3 or B9320-NIH3T3.In order to study the growth promoting function of B7032N or B9320, we with 4 kinds independently the B7032N-NIH3T3 cell (B7032N-A ,-B ,-C and-D) and 3 kinds of simulation-NIH3T3 cell (simulation-A independently,-B,-C), or 4 kinds of B9320-NIH3T3 cell (B9320-1 ,-2 independently,-3) and 3 kinds independently simulation-NIH3T3 cell (simulation-1,-2,3), inoculated 5 * 10 separately ³Individual cell, and (Japan) specification sheets by manufacturer carried out cell counting every day, through 5 days for DOJINDO, Kumamoto to adopt Cell-counting kit-8.These experiments are by carrying out in triplicate.

Make up C6700, B7032N and B9320 specific siRNA expression vector with psiU6X3.0

We are according to previous report (WO2004/076623; Shimokawa T, et al.Cancer Res.2003; 63:6116-20.) set up RNAi system with psiU6BX siRNA expression vector (to C6700) and PsiH1BX siRNA expression vector (to B7032N and B9320) based on carrier.By having prepared siRNA expression vector (psiU6BX-C6700, psiH1BX-B7032N or psiH1BX-B9320) in the BbsI site of the double chain oligonucleotide in the table 6 being cloned into the psiU6BX carrier at C6700, B7032N and B9320.Control plasmid: psiU6BX-simulation, Luc, out of order and EGFP is by preparing in the BbsI site of double chain oligonucleotide being cloned into the psiU6BX3.0 carrier.

The gene silencing effect of C6700, B7032N and B9320

RCC clone OS-RC-2 (being used for C6700), RXF-631L and A498 (being used for B7032N) and Caki-2 and A498 (being used for B9320) are plated on 10cm culture dish (1X10 ⁶Cell/ware), use FuGENE6 reagent (Roche), use Lipofectamine 2000 (Invitrogen) transfection psiU6BX-C6700, psiU6BX-B7032N and psiU6BX-B9320 and by the recommendation of provider as psiU6BX-simulation, Luc, the out of order and EGFP of negative control.After every kind of construct transfection 10 days,, use the low effect of striking of confirming siRNA at the Auele Specific Primer in the total zone of above-mentioned C6700 by sxemiquantitative RT-PCR then from the total RNA of cell extraction.Containing 0.7mg/ml Xin Meisu (Geneticin; Gibco BRL, Carlsbad, the OS-RC-2 cell of selection transfection in substratum CA).Then, after Geneticin is selected 5 days,, re-use following Auele Specific Primer group is measured siRNA by sxemiquantitative RT-PCR the low effect of striking from the total RNA of cell extraction:

As internal contrast: forward, 5 '-AACTTAGAGGTGGGGAGCAG-3 ' (SEQ IDNO:85) and oppositely, 5 '-CACAACCATGCCTTACTTTATC-3 ' (SEQ ID NO:86);

For C6700:5 '-TGACCTGTGCTTAGAAGTCCTTT-3 ' (SEQ ID NO:11) and 5 '-GTGTGTGTTCCTGAACAGATGAA-3 ' (SEQ ID NO:12);

For B7032N: forward, 5 '-CGGTGGTCTCTCATCCTTGT-3 ' (SEQ ID NO:101) and oppositely, 5 '-GCAACTCCTAACTCCCCTCTGTA-3 ' (SEQ ID NO:87);

For B9320: forward, 5 '-GCCTCGAGGTTATGCTTGAA-3 ' (SEQ IDNO, 102) and oppositely, 5 '-ATGCAGTCACTCACGCTCAG-3 ' (SEQ ID NO, 103).

At 3-(4,5-dimethylthiazole-2-yl)-2, during 5-phenylbenzene tetrazolium bromide (MTT) was measured, (DOJINDO, Kumamoto Japan) estimated cell viability according to supplier's rules to use Cell-counting kit-8 after 7 days and 14 days in transfection.Behind the incubation 21 days, fix these cells, and dye, to carry out colony forming assay with Giemsa solution with 4% Paraformaldehyde 96.

The result

Generally raise among the RCC or the evaluation of down-regulated gene

In order to prevent mixing of non-cancerous cells or improper cell, carried out the laser microbeam micro-dissection, on every side only to select the cancer cells and the cell that is derived from normal renal cortical cell in the tumor mass.As shown in Figure 1, the typical example of RCC micro-dissection from each clinical samples.Like this, we comparatively exactly (preciously) obtain the gene expression pattern of back.

For the mechanism on the oncogenesis basis of illustrating RCC, at first investigated and generally raised among the RCC or the gene of downward modulation.By the gene expression pattern among the 15 routine RCC, identified and expressed 972 genes that generally change; There are 251 genes in surpassing 50% information case, to show expression ratio, and compare, have 721 expression of gene in the RCC cell to be reduced to less than 0.2 (table 5) with normal renal cortical cell greater than 5.0 (referring to materials and methods) (tables 4).Comprise in these up-regulated genes be reported among the RCC cyclin-D1 (CCND1) of crossing high expression level (Hedberg Y., et al., (1999) Int.J.Cancer, 84:268-72.).This gene was high expression level in the present invention in 9 examples of 15 routine information cases.In these up-regulated genes, the biological function that 182 genes are arranged is known to a certain degree.In them, HIG2 (hypoxia inducible gene 2), NNMT (NNMT), IGFBP3 (insulin-like growth factor binding protein 3), VEGF (vascular endothelial growth factor) has been reported as with tumor of kidney with VWF (the VonWillebrand factor) and has formed relevant gene (Togashi A, et al., (2005) Cancer Res.; 65:4817-26; Yao M, et al., (2005) J Pathol.; 205:377-87; Cheung CW, et al., (2004) Kidney Int.; 65:1272-9; Staehler M, et al., (2005) CurrDrug Targets.; 6:835-46; Braybrooke JP, et al., (2000) Clin CancerRes.; 6:4697-704.), this has supported our microarray data high-quality.These up-regulated genes show various functions, comprise that (ADORA3 EDA2R), perhaps participates in multiple pathways metabolism (SCD to the signal transduction pathway genes involved, ENPP3), movement system (SLC1A3, ABCG1), the gene of vasculogenesis (VEGF), apoptosis (FTS) and cell adhesion (CDH2).Particularly, compare with normal cortex cell, have 92 expression levels to exceed in these up-regulated genes and surpass 10 times, for example ADORA3 (adenosine A 3 receptor), SLC1A3 (solute carrier family 1, the member 3) and STC2 (bony fish calsequestrin 2) raise above 10 times in surpassing 90% information case.532 in these down-regulated genes have obtained function to a certain degree and have characterized.This is comprising WT1 (wilms' tumor 1), CDKN1C (cell cycle protein dependent kinase inhibitor 1C) and GAS1 (cessation of growth cessation specificity 1), someone has hinted their effect (Niu Z, et al., (2005) JUrol in growth-inhibiting or apoptosis; 174:1 460-2; Kikuchi T, et al., (2002) Oncogene.; 21:2741-9; Watanabe H, et al., (1998) Proc Natl Acad Sci USA.; 95:1392-7; Evdokiou A ﹠amp; Cowled PA. (1998) Int J Cancer.; 75:568-77.).Particularly WT1 (wilms' tumor 1) and CDKN1C significantly downward modulation in 15 whole routine RCC cases, also significantly downward modulation in 14 examples of 15 examples of GAS1 shows that the downward modulation of these genes may be relevant with the generation of RCC tumour.

Up-regulated gene among table 4 RCC

Numbering	LMMID	Accession number	Symbol	Note
						1	F0661	AY048757	ABCG1	ATP-binding cassette, subfamily G (WHITE), the member 1
2	C4404	NM_052947	ALPK2	Alpha-kinases 2
					3	B8656	AY260577	C14orf58	Karyomit(e) 14 open reading-frame (ORF)s 58
4	B3940	K02765	C3	Complement component						3
					5	F0981	AK021624	C5orf13	Karyomit(e) 5 open reading-frame (ORF)s 13
6	A0206	M73554	CCND1	Cyclin D1 (PRAD1: parathyroid adenomatosis 1)
					7	E0525	BM988473	CCND1	Cyclin D1 (PRAD1: parathyroid adenomatosis 1)
8	C8039	Z22970	CD163	CD163 antigen
					9	B8048	BQ448718	CDC2L6	Cell division cycle 2 samples 6 (CDK8 sample)
10	A0692	X57548	CDH2	Cadherin						2, type 1, N-cadherin (neuronic)
					11	F2312	U57962	CG018	Hypothetical gene CG018
12	A5065	BC036661	CMKOR1	Chemokine orphan receptor (orphan receptor) 1
					13	F2133	AJ224864	CMRF-35H	The white corpuscle membrane antigen

14	D9371	H83622	COMMD10	Contain COMM territory 10
					15	A3153	M57730	EFNA1	Liver is joined albumen-A1
16	F8999	AK025273	EGLN3	Egl nine homologue 3 (Caenorhabditis elegans (C. elegans))
					17	A2065N	AK124656	ENO2	Hydratase, phosphoenolpyruvate 2 (gamma, neuronic)
18	C7756	H03641	FAM13A1	Has the homophylic family 13 of sequence, member A1
					19	A0797	J04162	FCGR3B	The Fc fragment of IgG, low-affinity IIIb, acceptor (CD16)
20	B8373	R27957	FCHO2	Only contain FCH territory 2
					21	E0847	NM_024830	FLJ12443	Hypothetical protein FLJ12443
22	F3997	AL049987	FLJ40092	FLJ40092 albumen
					23	A2464	U63917	GPR30	G protein coupled receptor 30
24	F6022	AK022479	HDHD1A	Contain hydracid dehalogenation enzyme sample lytic enzyme territory 1A
					25	C8023	M81141	HLA-DQB1	Main histocompatibility complex, II class, DQ beta 1
26	A1710	NM_002133	HMOX1	Heme oxygenase (unlinking) 1
					27	A1803	BC000013	IGFBP3	Insulin-like growth factor binding protein 3
28	A3181	NM_002193	INHBB	Statin, beta B (activator AB beta polypeptide)
					29	A7770	R55185	IRX3	Iroquois homology frame albumen 3
30	A2404	M15395	ITGB2	Integrin, beta 2 (antigens c D18 (p95), LFA 1; Scavenger cell antigen 1 (mac-1) beta subunit)
					31	B8882	BC005832	KIAA0101	KIAA0101
32	F2379	AB002365	KIAA0367	KIAA0367
					33	C5287	N91945	KIAA0746	KIAA0746 albumen
34	C7200	AB058765	KIAA1862	KIAA1862 albumen
					35	D7516	AI074524	LRRK2	Rich leucine tumor-necrosis factor glycoproteins kinases 2
36	A6527	T40467	MAPRE2	Microtubule-associated protein, RP/EB family, the member 2
					37	F8140	AW976457	MBNL1	Flesh blind (muscleblind) sample (fruit bat (Drosophila))
38	C4971	BC000234	NNMT	NNMT
					39	E0657	CR600491	NOL3	P120 3 (apoptosis repressor) with CARD territory
40	A4224	BC045651	P2RY5	Purinergic receptor P2Y, the G albumen coupling, 5
					41	A1757	M24486	P4HA1	Precollagen-proline(Pro), 2-oxoglutaric acid 4-dioxygenase (proline-4-hydroxylase), alpha polypeptide I
42	C6771	NM_002610	PDK1	Pyruvic dehydrogenase kinase, isoazyne 1
					43	A4010	BC002536	PFKP	Phosphofructokinase, thrombocyte
44	E1191	AA187749	PFKP	Phosphofructokinase, thrombocyte
					45	A5123	NM_000292	PHKA2	The phosphorylase kinase kinase enzyme, alpha 2 (liver)
46	A4486	AF059617	PLK2	Polo sample kinases 2 (fruit bat)
					47	A6551	AA811043	RNASET2	Ribonuclease T2
48	A4132	NM_005063	SCD	(delta-9-goes to satisfy stearyl-CoA desaturase

				And enzyme)
					49	A5240	BQ027924	SLC15A4	Solute carrier protein family 15, the member 4
50	E0623	AL162079	SLC16A1	Solute carrier protein family 16 (monocarboxylate transporter albumen), the member 1
					51	E1497	BU625507	SLC16A3	Solute carrier protein family 16 (monocarboxylate transporter albumen), the member 3
52	F2465	U88878	TLR2	Tol1 sample acceptor 2
					53	B8387	BC007372	TRIM52	Contain three gang mould bodies 52
54	A0478N	BC065522	VEGF	Vascular endothelial growth factor
					55	A3412	NM_000552	VWF	The Von Willebrand factor
56	F9001	AL137588	ZNF395	Zinc finger protein 39 5
					57	A5918	BX648117	ZNF6	Zinc finger protein 6 (CMPX1)
58	B6343	T79802		MRNA total length insertion sequence cDNA clones EUROIMAGE1534000
					59	B0741	BM991954		The seat of being transcribed faintly is similar to XP_311626.1 anopheles costalis (Anopheles gambiae str.PEST) ENSANGG00000020300 gene
60	B6492	AK057151		CDNA FLJ32589 fis, clone SPLEN2000443
					61	F7562	AI146812		Qb92e02.x1 Soares_ fetus _ heart _ NbHH19W people cDNA clones IMAGE:17075783 ', mRNA sequence.
62	F8824	AI916271		The seat of being transcribed
					63	E1182	CR599886	ADSL	The adenylosuccinate lyase
64	A8343	AK123519	ARHGAP26	RhoGTP enzyme activation protein 26
					65	C0227	N49962	BCL2	B-cell CLL/ lymphoma 2
66	C8205	BX648568	CARD8	Caspase is raised territory family, and the member 8
					67	F0691	NM_004932	CDH6	Cadherin	6, type 2, K-cadherin (fetal kidney)
68	C1993	AA831021	CLK4	CDC sample kinases 4
					69	A6942	AA521342	DDX31	DEAD (Asp-Glu-Ala-Asp) frame polypeptide 31
70	B6050	BG621779	EGFR	EGF-R ELISA (EBL virus (v-erb-b) oncogene homologue, birds)
					71	A8639	AI368204	ENPP3	The outer Nucleotide Pyrophosphate phosphohydrolase/phosphodiesterase 3 of born of the same parents
72	F0200	AL832950	FLJ31033	Hypothetical protein FLJ31033
					73	D0587	AA872040	INHBB	Statin, betaB (activator AB beta polypeptide)
74	A8946	AY423763	KLHL17	Kelch sample 17 (fruit bat)
					75	A9042	NM_022349	MS4A6A	Stride film 4-territory, subfamily A, member 6A
76	C6251	AA904502	MSL3L1	Male specificity						3 samples 1 (fruit bat) that cause death
					77	A6149N	NM_018092	NETO2	Neuropil albumen (NRP) and tolloid (TLL) sample 2
78	A1815	NM_002664	PLEK	Pleckstrin
					79	A8911	AK095793	PRKXP1	Protein kinase, X is chain, pseudogene 1

80	C9471	AK090411	RGPR	Regucalcin gene promoter area associated protein
					81	C6700	BC077726	SEMA5B	The Sema territory, seven thrombospondin tumor-necrosis factor glycoproteinss (1 type and 1 type sample), membrane-spanning domain (TM) and short cell matter territory, (brain signal albumen) 5B
82	B8658	CA429220	SKP2	S-phase kinase-associated protein 2 (p45)
					83	F0306	NM_003044	SLC6A12	(the neurotransmitter translocator, trimethyl-glycine/GABA), the member 12 in solute carrier protein family 6
84	B8485	CA308403	WIPI49	49kDa with the interactional WD40 repeat sequence protein of phosphoinositide
					85	E1336	XM_040486	ZMAT1	Zinc refers to, stromatin type 1
86	B0756	AA653431		The seat of being transcribed
					87	C7780	AA159605		The seat of being transcribed
88	D7481	BX392279		The seat of being transcribed is similar to the Buster3 transposase sample [people] that the XP_496781.1 transposon is originated consumingly
					89	D7317	AI015709		The seat of being transcribed
90	D8458	AA830668		Oc52h12.s1 NCI_CGAP_GCB1 people cDNA clones IMAGE:1353383 3 ', mRNA sequence
					91	D8466	AI619500		The seat of being transcribed
92	A0707	NM_000677	ADORA3	Adenosine A 3 receptor
					93	F2294	AK024900	AP2B1	Adapter (adaptor) associated protein mixture 2, beta 1 subunit
94	D3390	AL832660	C7orf29	Karyomit(e) 7 open reading-frame (ORF)s 29
					95	D9553	AL832896	CAPN12	Calpain 12
96	A1150	NM_000560	CD53	CD53 antigen
					97	F5859	AK023507	CMKOR1	Chemokine orphan receptor 1
98	F2310	AB002367	DCAMKL1	Dual cortin (doublecortin) and CaM kinases sample 1
					99	C2017	H88117	DKFZP564J102	DKFZP564J102 albumen
100	B0726	BC034919	EDA2R	Ectodermal dysplasia albumin A 2 acceptors
					101	C8054	NM_001445	FABP6	Fatty acid binding protein 6, (gastrotropin) of ileum
102	A1051	BM662950	FCER1G	The Fc fragment of IgE, high-affinity I, acceptor; The gamma polypeptide
					103	B7439	N51406	FLJ14503	Hypothetical protein FLJ14503
104	B9480	AB018345	KIAA0802	KIAA0802
					105	F6208	XM_058513	LRRK2	Rich leucine tumor-necrosis factor glycoproteins kinases 2
106	E1387	D87448	topBP1	Topoisomerase (DNA) II conjugated protein 1
					107	A9518N	AA570186		Hypothetical gene by the AK096951 support; BC066547
108	F5778	AK021801		CDNA FLJ11739 fis, clone HEMBA1005497

109	C4457	BM665350	AIG1	Male sex hormone inductive 1
					110	A2811	X00570	APOC1	ApoC-I
111	C9189	BC065544	C14orf106	Karyomit(e) 14 open reading-frame (ORF)s 106
					112	B8942	AA868809	CHC1L	Karyomit(e) concentrates 1 sample
113	A5716	U78556	CRA	Cis-platinum (Cisplatin) resistance is correlated with
					114	A1687	U29171	CSNK1D	Casein kinase 1, delta
115	D3230	AA780074	CSS3	Chondroitin sulfate synthase 3
					116	C2022	H88362	EHBP1	EH territory conjugated protein 1
117	C7370	BC037568	EOMES	Eomesodermin homologue (xenopous laevis (Xenopus laevis))
					118	A2115	BQ949386	FCGR1A	The Fc fragment of IgG, high-affinity Ia, acceptor (CD64)
119	C6584	BC042143	FLJ32009	Hypothetical protein FLJ32009
					120	A2839	M36284	GYPC	Glycophorin C (Gerbich blood group)
121	A2715	BC035802	GZMK	Granzyme K (serine protease, granzyme 3; Tryptase II)
					122	A3113	M60445	HDC	L-Histidine decarboxylase.
123	D9621	NM_178229	IQGAP3	The GTP enzyme activation albumen 3 that contains the IQ die body
					124	D3104	AK091335	MARCH-I	The RING-CH protein I that film is relevant
125	A2753N	BC009924	NPTX2	Neural five poly-cyclase protein II
					126	B7032N	AA398096 NM_004567	PFKFB4	6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 4
127	C9099	BM724257	PLEKHM2	Contain the pleckstrin homeodomain, the M of family (having the RUN territory) member 2
					128	B8547	BC033746	PNCK	The CaM kinases that the non-omnipresence that gestation raises is expressed
129	F3374	AF195765	RAMP	The nuclear matrix associated protein that RA regulates
					130	A4587	AF070609	SLC1A3	DKFZP547J0410 albumen
131	B4440	AB040120	SLC39A8	Solute carrier protein family 39 (zinc translocator), the member 8
					132	B4578	AI290343	STC2	Department's gland calsequestrin (Stanniocalcin) 2
133	A1064	NM_024164	TPSB2	Tryptase alpha/beta 1
					134	B8105	AI023320		The LOC387790 that supposes
135	E0400	N46844		CDNA:FLJ20892fis, clone ADKA03430
					136	B7272	BQ268701		Ir34g09.x1HR85 pancreas islet (islet) people cDNA clones IMAGE:65472173 ', mRNA sequence
137	B1075	BU623171		C DNA cloning IMAGE:3029742, part cds
					138	F5749	AK025204	ABI3BP	The ABI gene family, member 3 (NESH) is conjugated protein
139	B8361	AI741514	ALPK2	Alpha-kinases 2
					140	A4505	S60415	CACNB2	Calcium channel, voltage relies on, beta 2 subunits

141	F2918	AF376061	CARD12	Caspase is raised territory family, and the member 12
					142	A3560	L06797	CXCR4	Chemokine (C-X-C die body) acceptor 4
143	B7437	AK024965	DAB2	The homologue of loss of function (disabled homolog) 2, mitogen responsiveness phosphorprotein (fruit bat)
					144	B1960	AI821270	DARS	Aspartyl-tRNA synthetase
145	D9765	AA442590	ENPP3	The outer Nucleotide Pyrophosphate phosphohydrolase/phosphodiesterase 3 of born of the same parents
					146	B9275	BU736022	FABP7	Fatty acid binding protein 7, brain
147	A6168	AK123603	FLJ12443	Hypothetical protein FLJ12443
					148	A9014	AK023320	FTS	Merge toe homologue (mouse)
149	B1834	AW978675	GNRH1	Gonadotropin-releasing hormone 1 (luteinization-releasing hormone)
					150	F2358	AK021481	GPC6	Glypican 6
151	C0773	BX537890	KLF7	The similar factor 7 of Kruppel (omnipresence)
					152	B6517	BC024278	LOC255326	Hypothetical protein LOC255326
153	F3337	AK026747	LOC54103	Hypothetical protein LOC54103
					154	D5370	AA907927	MDS009	X 009 albumen
155	D4999	AA971400	MGC47816	Hypothetical protein MGC47816
					156	E0087	BX484485	MLL3	B melanoma antigen family, the member 4
157	B8611	W60419	POLI	Polysaccharase (at DNA's) iota
					158	A6389	BC084567	POLS	Polysaccharase (at DNA's) sigma
159	C2006	H87865	RFP2	Ret finger protein 2
					160	A0949	NM_005505	SCARB1	The scavenger receptor category-B, the member 1
161	B3918	AK027663	STC2	Department's gland calsequestrin (Stanniocalcin) 2
					162	F3366	AL080215	TFPI	Tissue factor approach restrainer (the blood coagulation inhibitor that lipoprotein is relevant)
163	A6906	BC050423	TMEM22	Transmembrane protein 22
					164	C4733	AA342991		(iPABP) (activatory-blood platelet albumen-1) (APP-1) to be similar to polyadenylic acid-conjugated protein 4 (Poly (A)-conjugated protein 4) (PABP 4) (derivable poly (A)-conjugated protein)
165	D7225	AA827683		The seat of being transcribed, moderate is similar to NP_775735.11 (3) mbt sample 4 (fruit bat) [people]
					166	C1701	H60869		Yr46b12.s1Soares fetal livers spleen 1NFLS people cDNA clones IMAGE:2083193 ', mRNA sequence.
167	D9545	AI076475		Hypothetical gene by the AK026189 support
					168	C0801	AA205305	ANKRA2	Ankyrin repeat, the A of family (RFXANK sample), 2
169	A8990	NM_013448	BAZ1A	Refer to the Bu Luomo structural domain that the territory is adjacent, 1A with zinc
					170	B9465	BC039999	C9orf76	Karyomit(e) 9 open reading-frame (ORF)s 76
171	A6965	R78576	COG6	The component 6 of oligomerization Golgi complex

172	F3077	NM_001003927	EVI2A	Ecotropic virus integration site 2A
					173	B9320	BC036680 BC034014 NM_153350	FBXL16	The protein 16 of rich F-frame and leucine tumor-necrosis factor glycoproteins
174	C9574	Y18046	FGFR1OP	FGFR1 oncogene mating partner
					175	A6659	AK096712	FLJ11850	Hypothetical protein FLJ11850
176	F4018	AK022473	FTO	Fatso
					177	C7773	AF430643	GBP5	Guanine nucleotide binding protein 5
178	B7466	AA128378	KIAA0303	The serine/threonine kinase family member 4 that microtubule is relevant
					179	A3867	AF013249	LAIR1	The Ig sample acceptor 1 that white corpuscle is relevant
180	C5174	AL832259	LOC284749	Hypothetical protein LOC284749
					181	D3452	BX482647	PARP14	Poly-(ADP-ribose) polysaccharase family, the member 14
182	B1210	AA398762	PHKA2	The phosphorylase kinase kinase enzyme, alpha 2 (liver)
					183	A3613	CR608325	PLA2G7	Phospholipase A2, VII organizes (thrombocyte-activation factor PAF-AH, blood plasma)
184	A2498	L11932	SHMT2	Serine hydroxymethylase 2 (mitochondrial)
					185	E1370	NM_000544	TAP2	Transport protein 2, ATP-binding cassette, subfamily B (MDR/TAP)
186	B7455N	BX112530	TLK1	Tousled sample kinases 1
					187	F4886	AK026403	TLN2	Talin (Talin) 2
188	A0504	NM_005428	VAV1	Vav 1 oncogene
					189	C6417	BU742664		Total length insertion sequence cDNA clones ZE12B03
190	C1799	BQ007156		The seat of being transcribed
					191	C4116	XM_496823		Be similar to RIKEN cDNA A630077B13 gene; RIKEN cDNA 2810048G17
192	B0732	AA632745		The seat of being transcribed
					193	B8870	NM_018685	ANLN	Born of the same parents' cyclase protein, actin binding protein (scraps homologue, fruit bat)
194	D3310	NM_019043	APBB1IP	Amyloid beta (A4) precursor protein associativity, the B of family, member's 1 interaction albumen
					195	B3966	BC047724	C10orf128	Karyomit(e) 10 open reading-frame (ORF)s 128
196	B3130	BQ025155	C10orf9	Karyomit(e) 10 open reading-frame (ORF)s 9
					197	C4454	AK026746	C14orf140	Karyomit(e) 14 open reading-frame (ORF)s 140
198	C0579	BX648468	DKFZP564J086 3	DKFZP564J0863 albumen
					199	B7451N	AY358175	GBGT1	Globoside alpha-1,3-N-acetylgalactosamine based transferase 1
200	F3828	U79242	GRM5	Glutamate receptor, metabolic pattern 5
					201	A2681	NM_001884	HAPLN1	Hyaluronan is connected albumen 1 with proteoglycan
202	A6379	BM464532	HIST1H1C	Histone 1, H1c
					203	F0266	NM_000878	IL2RB	The interleukin II acceptor, beta
204	F6738	AK022173	LAF4	The lymphocyte nucleoprotein relevant with AF4

205	A8928	R38549	LOC150271	The LOC388889 that supposes
					206	C7670	AA156409	MCPH1	The microcephaly, primary autosomal recessive 1
207	C5756	AK090397	MGC15875	Hypothetical protein MGC15875
					208	C6664	AI142832	MGC34923	Hypothetical protein MGC34923
209	D8440	AA826148	NRCAM	The neuronal cell adhesion molecule
					210	A3896	BC015050	OIP5	Opa-interaction albumen 5
211	C9148	AA282540	OSBPL8	The conjugated protein sample 8 of oxygen sterol (oxysterol)
					212	E1852	AA258620	PLXNC1	Clump PROTEIN C 1
213	F0887	AK021778	PTPRK	Protein-tyrosine-phosphatase, receptor type, K
					214	B9282	R60655	RNF32	Ring finger protein 32
215	B7289N	AF146761	SLAMF8	SLAM family member 8
					216	F3858	AB020701	SORBS1	Contain sorbose and SH3 territory 1
217	F4051	M31165	TNFAIP6	Tumour necrosis factor, alpha inductive albumen 6
					218	C2049	N24715		Total length insertion sequence cDNA clones YX74D05
219	C8379	W57613		The seat of being transcribed faintly is similar to NP_062553.1 hypothetical protein FLJ11267 [people]
					220	C9252	AK126261		Be similar to RIKEN cDNA E030024N20 gene
221	D2965	BU622474		Be similar to D (1B) Dopamine Receptors (D (5) Dopamine Receptors) (D1beta Dopamine Receptors)
					222	C4328	AK023966		CDNA FLJ13904fis, clone THYRO1001895
223	A2677	Z11502	ANXA13	Annexin A13
					224	C3760	BC008718	BIRC5	Contain baculovirus IAP tumor-necrosis factor glycoproteins 5 (Survivin)
225	B3180N	BM992179	CYB5	Cytochrome b-5
					226	C8944	AK001149	FLJ10287	Hypothetical protein FLJ10287
227	C7777	BU191922	FLJ23861	Hypothetical protein FLJ23861
					228	B2874	AA883488	KIAA0408	KIAA0408
229	B8940	BX641066	KLF8	The similar factor 8 of Kruppel
					230	D5261	BC033490	LOC285016	Hypothetical protein LOC285016
231	D5688	AL832281	LOC340351	Hypothetical protein LOC340351
					232	D3549	BU620736	MAGI-3	Relevant (MAGI-3) of guanylate kinase that film is relevant
233	A8335	BC028421	MGC33630	Hypothetical protein MGC33630
					234	F3431	AK021954	NRCAM	The neuronal cell adhesion molecule
235	D7468	BC010943	OSMR	The carcinogenic protein m receptor
					236	B7110	AK124752	PCDH21	Former cadherin 21
237	C0488	AA781195	PRAME	Preferential antigen of expressing in the melanoma
					238	C8373	R77952	PSMA3	Proteasome (prosome, macropain) subunit, alpha type, 3
239	D3765	AK096798	RDH13	Retinol dehydrogenase 13 (alltrans and 9-cis)
					240	B6688	NM_003042	SLC6A1	Solute carrier protein family 6 (neurotransmitter transhipment

				Albumen, GABA), the member 1
					241	A5720	BQ787632	SPON1	Spondin	1, extracellular matrix protein
242	B9009	AB011123	TNIK	TRAF2 and NCK interaction kinases
					243	B0869N	AF274048	UHRF1	The ubiquitin sample contains PHD and fourth finger territory, 1
244	C4641	BF115786	ZCCHC11	Zinc refers to, contains CCHC territory 11
					245	C8919	CR749811		Be similar to hypothetical protein MGC38937
246	C6314	W86513		The seat of being transcribed faintly is similar to XP_517256.1UDP glycosyltransferase 2 families, polypeptide B4[chimpanzee (Pan troglodytes)]
					247	A9641	BG196354		The seat of being transcribed
248	D5230	AA938691		The seat of being transcribed
					249	D1218	BQ709650		The seat of being transcribed
250	B1929	AA663031		Ab72b05.s1 Stratagene fetal retinal 937202 people cDNA clone IMAGE:852465 3 ', mRNA sequence.
					251	D6557	AK092260		CDNA FLJ34941fis, clone NT2RP7007480

Down-regulated gene among the table 5RCC

Numbering	LMMID	Accession number NO	Symbol	Note
					252	C6486	X83618	HMGCS2	3-hydroxy-3-methylglutaric acid list acyl-coenzyme A synthase 2 (mitochondrial)
253	A4297	NM_012205	HAAO	3-hydroxyanthranilic acid 3, the 4-dioxygenase
					254	A1767	M93107	BDH	3-hydroxybutyric dehydrogenase (heart, mitochondrial)
255	A2694N	AK057510	HPD	4-hydroxyphenyl pyruvic acid dioxygenase
					256	B3053	AI732791	XRN1	5 '-3 ' exoribonuclease 1
257	D4971	AA918686	PFKFB2	6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 2
					258	A6530	NM_006988	ADAMTS1	A with thrombospondin type 1 die body de-connects albumen sample and metalloprotease (reprolysin type), 1
259	B3397	AA732989	ADAMTS2	A with thrombospondin type 1 die body de-connects albumen sample and metalloprotease (reprolysin type), 2
					260	A1182	U23435	ABI2	Abl interaction factor (interactor) 2
261	B8954	NM_032432	ABLIM2	Actin muscle associativity LIM protein family, the member 2
					262	A1850	L22214	ADORA1	Adenosine A	1 receptor
263	F0673	AF062595	AK5	Myokinase							5
					264	C7731	AF245505	DKFZp564I1922	Adlican
265	B3219	T67409	AFM	Afamin
					266	A3322	M80899	AHNAK	AHNAK nucleoprotein (desmoyokin)

267	A2323	V00494	ALB	White protein
					268	A2644	BC062476	ADH1C	Alcoholdehydrogenase 1C (I class), the gamma polypeptide
269	A1760	BC039065	ADH6	Alcoholdehydrogenase 6 (V class)
					270	A2508	X03350	ADH1B	Alcoholdehydrogenase IB (I class), the beta polypeptide
271	A5088	U24266	ALDH4A1	Aldehyde dehydrogenase 4 families, member A1
					272	A3081	NM_005589	ALDH6A1	Aldehyde dehydrogenase 6 families, member A1
273	A2056	X02747	ALDOB	ALD-B, fructose-bisphosphate
					274	A2084	BM709336	AIF1	Allotransplantation inflammatory factor 1
275	B3363	AA806986	ATRX	Alpha thalassemia/backwardness syndrome X chain (RAD54 homologue, yeast saccharomyces cerevisiae (S.cerevisiae)
					276	B3404	NM_015120	ALMS1	Alstrom syndrome 1
277	B5424	NM_020039	ACCN2	Guanamprazine (Amiloride) susceptibility cationic channel 2, neuronic
					278	A4409	NM_005503	APBA2	Amyloid beta (A4) precursor protein associativity, the A of family, member 2 (X11 sample)
279	B9341	BC012984	ALS2CR19	Amyotrophic lateral sclerosis 2 (teenager) chromosomal region, the candidate 19
					280	F3692	NM_004673	ANGPTL1	Vascularization element (Angiopoietin) sample 1
281	A3360	NM_031850	AGTR1	Angiotensin-ii receptor, 1 type
					282	B5155	W84893	AGTRL1	Angiotensin-ii receptor sample 1
283	A9039	AK095632	ABTB2	Contain ankyrin repeat and BTB (POZ) territory 2
					284	C7993	NM_020349	ANKRD2	Ankyrin repeat territory 2 (the reactive muscle of stretching)
285	A3006	M62839	APOH	Apolipoprotein H (beta-2-glycoprotein I)
					286	A4483	AF049884	ARGBP2	Arg/Abl-interaction albumin A rgBP2
287	A2008	BC029050	ARG2	Arginase, the II type
					288	B4126	AK027126	ASS	Argininosuccinate synthetase
289	A3010	NM_080912	ASGR2	Asialoglycoprotein acceptor 2
					290	C7318	BM677716	ATP8A2	The ATP enzyme, amino phosphatide translocator sample, the I class, type 8A, the member 2
291	A3380	L20977	ATP2B2	The ATP enzyme, Ca++ transhipment property, plasma membrane 2
					292	D5516	AI261591	ATP6V1G3	The ATP enzyme, H+ transhipment property, lysosome 13kDa, V1 subunit G isotype 3
293	A0859	M25809	ATP6V1B1	The ATP enzyme, H+ transhipment property, lysosome 56/58kDa, V1 subunit B, isotype 1 (following deaf renal tubular acidosis)
					294	A5628	NM_001690	ATP6V1A	The ATP enzyme, H+ transhipment property, lysosome 70kDa, V1 subunit A
295	D1054	AA994979	ATP6V0A4	The ATP enzyme, H+ transhipment property, lysosome V0 subunit a isotype 4
					296	A3090	BC031609	ATP12A	The ATP enzyme, H+/K+ transhipment property, non-stomach, alpha polypeptide

297	C3735	NM_000701	ATP1A1	The ATP enzyme, Na+/K+ transhipment property, alpha 1 polypeptide
					298	F3709	NM_152296	ATP1A3	The ATP enzyme, Na+/K+ transhipment property, alpha 3 polypeptide
299	D5023	AK131204	BTLA	B is relevant with the T lymphocyte
					300	B5356	AA758171	MLL3	B melanoma antigen family, the member 4
301	A3079	J04599	BGN	Disaccharide catenin glycan
					302	A4481	AF053470	BLCAP	The albumen that bladder cancer is relevant
303	A1922	CR605506	BAMBI	The membrane-bound inhibitor homologue of BMP and activator (xenopous laevis (Xenopus laevis))
					304	A2545	BC033873	BST2	Marrow stromal cell antigen 2
305	A2691N	BC041846	CDH3	Cadherin 3,1 types, P-cadherin (placenta 1)
					306	A3072	NM 199248	CACNB1	Calcium channel, voltage relies on, beta 1 subunit
307	A3062	BC025687	CAMK4	Calcium/calmodulin-dependent protein kinase IV
					308	A4819	D17408	CNN1	Calmodulin 1, alkalescence, unstriated muscle
309	B5410	BC033183	CHST3	Sugar (chrondroitin 6) sulfotransferase 3
					310	A7810	BC022567	CHST1	Sugar (keratan sulfate Gal-6) sulfotransferase 1
311	A3324	BC057792	CA4	Carbonic anhydrase IV
					312	B0852	H23177	CA10	Carbonic anhydrase X
313	A1754	AB119995	CES1	Procaine esterase 1 (monocyte/macrophage serine easterase 1)
					314	C8606	BC063430	CPXM	Carboxypeptidase X (M14 family)
315	C4120	X66417	CSN3	Casein kappa
					316	A1912	BC052996	CTNNA2	Connection albumen (cadherin associated protein), alpha 2
317	A3822	BC067289	CTSL2	Cathepsin L 2
					318	C0791	BC051340	CD164L1	CD164 sialomucin sample 1
319	A5148N	NM_006566	CD226	CD226 antigen
					320	A3224	J04132	CD3Z	CD3Z antigen, zeta polypeptide (TiT3 mixture)
321	E0542	NM_152243	CDC42EP1	CDC42 effector albumen (RhoGTP enzyme associativity) 1
					322	A9130	AF001436	CDC42EP2	CDC42 effector albumen (RhoGTP enzyme associativity) 2
323	A6126N	H11384	CDC42EP3	CDC42 effector albumen (RhoGTP enzyme associativity) 3
					324	B6737	NM_012127	CIZ1	CDKN1A interaction zinc finger protein 1
325	C7260	BC071790		C DNA cloning IMAGE:4611512, part cds
					326	B4933	R12478		C DNA cloning IMAGE:4797817, part cds
327	C5860	BU683028		CDNA FLJ10151 fis, clone HEMBA1003402

328	F3736	AK021754		CDNA FLJ11692fis, clone HEMBA1004983
					329	B3217	AI279516		CDNA FLJ11723fis, clone HEMBA1005314
330	D1811	AK128814		CDNA FLJ25106fis, clone CBR01467
					331	D4748	AK055811		CDNA FLJ31249fis, clone KIDNE2005327
332	D3276	AA765718		CDNA FLJ33551fis, clone BRAMY2009105
					333	B3261	AK091765		CDNA FLJ34446fis, clone HLUNG2002050
334	C9234	AK093732		CDNA FLJ36413fis, clone THYMU2010816
					335	F1221	AL109700		CDNA FLJ37610fis, clone BRCOC2011398
336	C9594	N71087		CDNA FLJ37676fis, clone BRHIP2012627
					337	C4381	W45330		CDNA FLJ38345fis, clone FCBBF3028671
338	D6230	AA977738		CDNA FLJ40061fis, clone TESOP2000083
					339	B8241	AK075052		CDNA FLJ90571fis, clone OVARC1001725 highly is similar to people patched associated protein TRC8 (TRC8) gene.
340	F2149	AK024898		CDNA:FLJ21245fis, clone COL01184
					341	A5311N	BQ718658		CDNA:FLJ22642fis, clone HSI06970
342	B3296	BU741595	LHX4	LIM homology frame 4
					343	A6754	NM_004378	CRABP1	Cellular retinoic acid binding protein 1
344	E1369	AK025651	PNAS-4	CGI-146 albumen
					345	C6124	NM_002989	CCL21	Chemokine (C-C die body) part 21
346	A1660	BC048282	CLCNKA	Chloride channel Ka
					347	F1739	AF216941	CLIC5	Chlorine born of the same parents interior passageway 5
348	B2906	AI808724	CHRNB2	Cholinergic receptor, nicotine, beta polypeptide 2 (neural)
					349	C8926	BU569535	CHODL	The cartilage lectin
350	C8476	R59552	CHRDL1	Plain sample						1 takes place in notochord
					351	A3539	NM_001275	CHGA	Chromogranin A (parathyroid secretory protein 1)
352	A2596	BC000375	CHGB	Chromogranin B (secretogranin 1)
					353	A6346	NM_015576	C1orf1	Karyomit(e) 1 open reading-frame (ORF) 1
354	B6193N	NM_030806	C1orf21	Karyomit(e) 1 open reading-frame (ORF) 21
					355	B9135	BC066977	Clorf40	Karyomit(e) 1 open reading-frame (ORF) 40
356	F6114	AK025270	C10orf137	Karyomit(e) 10 open reading-frame (ORF)s 137
					357	F6052	BF967878	C10orf83	Karyomit(e) 10 open reading-frame (ORF)s 83

358	F4642	NM_001584	Cllorf8	Karyomit(e) 11 open reading-frame (ORF)s 8
					359	D3719	H08298	C13orf7	Karyomit(e) 13 open reading-frame (ORF)s 7
360	F7019	AK001590	C14orf132	Karyomit(e) 14 open reading-frame (ORF)s 132
					361	D6180	AK096674	C14orf32	Karyomit(e) 14 open reading-frame (ORF)s 32
362	F1846	AK023979	C14orf46	Karyomit(e) 14 open reading-frame (ORF)s 46
					363	F0168	BC015178	C18orf10	Karyomit(e) 18 open reading-frame (ORF)s 10
364	B4749	AA715708	C18orf49	Karyomit(e) 18 open reading-frame (ORF)s 49
					365	F6269	AY327407	C2orf10	Karyomit(e) 2 open reading-frame (ORF)s 10
366	D3266	AA617917	C20orf65	Karyomit(e) 20 open reading-frame (ORF)s 65
					367	B0294	BC029130	C6orf35	Karyomit(e) 6 open reading-frame (ORF)s 35
368	A6409	AK091288	C9orf19	Karyomit(e) 9 open reading-frame (ORF)s 19
					369	D1204	XM_059954	C9orf57	Karyomit(e) 9 open reading-frame (ORF)s 57
370	A5356	NM_001002260	C9orf58	Karyomit(e) 9 open reading-frame (ORF)s 58
					371	B0676	AK126055	C9orf84	Karyomit(e) 9 open reading-frame (ORF)s 84
372	B9368	AF504647		Cilium related protein (CYS1)
					373	C0661	H18687	CLDN11	Close protein 11 (oligodendrocyte transmembrane protein)
374	A2460	AF000959	CLDN5	Close albumen 5 (transmembrane protein that in velo-cardio-facial syndrome, lacks)
					375	A6080	N99340	CLIPR-59	The CLIP-170 associated protein
376	B8234	AF070632		Clone 24405mRNA sequence
					377	A5341	AF131834		Clone 24841mRNA sequence
378	C4665	AK022877		Clone TUA8Cri-du-chat district mRNA
					379	A2191	M16967	F5	Factor V (proaccelerin, instability factor (liable factor))
380	A3265N	NM_004291	CART	Cocaine and amphetamine modulability transcript
					381	B0268	AK123393	CCDC3	Contain the curling-territory 3 of curling
382	A3156	L02870	COL7A1	Collagen, VII type, alpha 1 (epidermolysis bullosa, dystrophic, dominance and recessiveness)
					383	C1520	BC014640	COL14A1	Collagen, type XIV, alpha 1 (undulin)
384	A3963	NM_080542	COLQ	The collagen sample afterbody subunit (tail subunit) (strand of homotrimer) of asymmetry acetylcholinesterase
					385	C0431	BX538105	COLM	Collomin
386	C7997	J03565	CR2	Complement component (3d/Epstein Barr virus) acceptor 2
					387	F2392	NM_001901	CTGF	Connective Tissue Growth Factor
388	B0555	AA533963	CPNE4	Copine IV
					389	A1793	X58022	CRHBP	Thyroliberin discharges sex hormone binding globulin
390	F2073	NM_020990	CKMT1	Creatine kinase, plastosome 1 (omnipresence)
					391	A1403	J05401	CKMT2	Creatine kinase, plastosome 2 (muscle segment)
392	A1171N	U05569	CRYAA	Crystallin, alphaA
					393	D8699	BX117062	CTBP2	C-terminal conjugated protein 2
394	A7291	BC042692	CTL2	The CTL2 gene

395	A0775	L12579	CUTL1	Cut sample 1, CCAAT replaces (displacement) albumen (fruit bat)
					396	D4370	AA883874	CXXC4	CXXC refers to 4
397	A0260	U47413	CCNG1	Cyclin G1
					398	A9564	NM_000076	CDKN1C	Cell cycle protein dependent kinase inhibitor 1C (p57, Kip2)
399	A2452	BX537488	CSRP1	Rich halfcystine and glycine albumen 1
					400	B7775	AF169802	CYB5R2	Cytochrome b5 reductase b5R.2
401	D6271	AY161004	COX8C	Cytochrome c oxidase subunit 8C
					402	D1185	AA451886	CYP1B1	Cytochrome P450, family 1, subfamily B, polypeptide 1
403	A0920N	NM_000773	CYP2E1	Cytochrome P450, family 2, subfamily E, polypeptide 1
					404	C7590	BC001776	CYP27B1	Cytochrome P450, family 27, subfamily B, polypeptide 1
405	D9075	AL832156	CPEB1	Tenuigenin polyadenylation element conjugated protein 1
					406	C7057	H22566	DACH1	Dachshund homologue 1 (fruit bat)
407	A4586	D86977	DHX38	DEAH (Asp-Glu-Ala-His) frame polypeptide 38
					408	A0922	NM_004394	DAP	Dead associated protein
409	C2154	AF007144	DIO2	Take off the iodine enzyme, iodate desiodothyroxine, II type
					410	B5367	BC033736	DPT	Dermatopontin
411	B5268	BQ062201	DCXR	Dicarbapentaborane/L-xyloketose reductase
					412	A2257N	BC052998	DDR2	Net handle bacterium coagulates the prime field receptor family, and the member 2
413	A0283	NM_021120	DLG3	Discs, big homologue 3 (neuroendocrine-dlg, fruit bat)
					414	A8605	NM_015393	DKFZP564O082 3	DKFZP564O0823 albumen
415	A2029	BC034227	D4S234E	Dna fragmentation on the karyomit(e) 4 (unique) 234 expressed sequences
					416	A0748	M76180	DDC	Dopa decarboxylase (aromatics L-amino acid decarboxylase)
417	A8098	AL713793	DAPP1	The dual adapter (adaptor) of Tyrosine O-phosphate and 3-phosphoinositide
					418	A0623	NM_001395	DUSP9	Dual specificity phosphatase enzyme 9
419	A3054	U01839	FY	The Duffy blood group
					420	A0192	M62829	EGR1	Early growth replys 1
421	E1348	BX640908	EVI1	Ecotropic virus integration site 1
					422	A4665	D12485	ENPP1	The outer Nucleotide Pyrophosphate phosphohydrolase/phosphodiesterase 1 of born of the same parents
423	B8411	BX412247	EFHD1	Contain EF hand shape territory 1
					424	C9877	BQ001493	EHD3	Contain EH-territory 3
425	B6831	NM_000118	ENG	Endothelium glycoprotein (Osler-Rendu-Weber syndrome 1)
					426	A7247N	AL133118	EMCN	Interior Saliva Orthana
427	D6367	NM_152461	ERN1	Endoplasmic reticulum is to nuclear signal conduction 1

428	A3097	M65199	EDN2	The endothelium blood vessel element 2 that contracts
					429	A8387	AL697428	ENTH	Enthoprotin
430	C0774	AK023744	EPS15L1	EGF-R ELISA approach substrate 15 samples 1
					431	A2019N	AA442410	EMP1	Epithelial membrane albumen 1
432	A8525	W67837	EMP2	Epithelial membrane albumen 2
					433	D4636	AF370395	EPS8L1	EPS8 sample 1
434	C2324	BQ182775	ECRG4	Esophageal cancer related gene 4 albumen
					435	A3160N	NM_000125	ESR1	Estrogen receptor 1
436	F3825	BC064700	ESRRG	Estrogen-related receptor gamma
					437	B5753	BC010082	ETNK2	Ethanolamine kinase 2
438	C7625	BU684240	EHF	The Ets homology factor
					439	A1553	BC023505	ECM1	Extracellular matrix protein 1
440	A4438	AF055015	EYA2	Anophthalmia homologue 2 (fruit bat)
					441	A7246	N75862	EYA4	Anophthalmia homologue 4 (fruit bat)
442	B7281	NM_058186	FAM3B	Has the homophylic family 3 of sequence, member B
					443	A8682	AL713770	FAM31C	Has the homophylic family 31 of sequence, member C
444	F0573	AB032996	FAM40B	Has the homophylic family 40 of sequence, member B
					445	B5086	CR595341	FLJ90024	Empty stomach induction type (fasting inducible) integral protein TM6P1
446	A0441	CR612744	FABP1	Fatty acid binding protein 1, liver
					447	A2542	J02874	FABP4	Fatty acid binding protein 4, adipocyte
448	B6097	W39428	FBXO2	F-frame albumen 2
					449	A5840N	AA738314	FBXO42	F-frame protein 42
450	A6380	NM_005141	FGB	Fibrinogen, B beta polypeptide
					451	A0075N	D14838	FGF9	Fibroblast growth factor 9 (HBFG-9)
452	B6082	BX537781	FNDC5	Contain fibronectin II type I territory 5
					453	A8531	BX537531	FBLN5	Fine albumen 5
454	A5736	AF052170	MICAL2	Flavoprotein oxydo-reductase MICAL2
					455	A9082	H77985	FLJ25476	FLJ25476 albumen
456	A5895	AL713743	FLJ42875	FLJ42875 albumen
					457	C9358	AI126777	FLJ45455	FLJ45455 albumen
458	A9984	AK057972	LOC51066	Fls485
					459	B5279	BC004107	FST	Follistatin
460	B3525	NM_012188	FOXI1	Forkhead frame I1
					461	F6422	AW964166	FTCD	The formimino transferase cyclodeaminase
462	B8503	AF225426	FMN2	Become albumen 2
					463	B4294	NM_012190	FTHFD	Formyltetrahydrofolate dehydrogenase
464	B2754	W94882	C9orf154	The extracellular matrix 1 that FRAS1 is relevant
					465	D9934	CA450275	FREQ	Frequenin homologue (fruit bat)
466	A0764	L10320	FBP1	Fructose-1 1
					467	C0328	BE080213		The full length cDNA clone of homo sapiens (people) placenta

				CS0DE011YI04
					468	D1157	AW977392		The normalized cDNA clone of homo sapiens (people) total length placenta Cot 25-CS0DI042YD07
469	B3229	AK026244	GPR107	G protein coupled receptor 107
					470	A4491	L15388	GRK5	G protein coupled receptor kinases 5
471	C6906	AK122672	GPCR5A	G protein coupled receptor, the C of family, 5 groups, member A
					472	A5937	BC028315	GABARAPL1	GABA (A) receptor associated protein(RAP) sample 1
473	A3352	NM_000807	GABRA2	Gamma-aminobutyric acid (GABA) A acceptor, alpha2
					474	A2418	M96789	GJA4	Gap junction protein, alpha4,37kDa (connection protein 37)
475	A5978	BQ003596	GJA5	Gap junction protein, alpha 5,40kDa (connecting albumen 40)
					476	A1859N	NM_001002295	GATA3	GATA conjugated protein 3
477	A0697	L20316	GCGR	Glucagon receptor
					478	A2059N	M81883	GAD1	L-Glutamic decarboxylase 1 (brain, 67kDa)
479	A6127	AI356291	GPT2	Gpt (alanine aminotransferase) 2
					480	A2801	NM_002083	GPX2	Selenoperoxidase 2 (stomach and intestine)
481	A1610	NM_002084	GPX3	Selenoperoxidase 3 (blood plasma)
					482	A4202	BC053578	GSTA1	Glutathione S-transferase A1
483	A2479	L34041	GPD1	Glycerol-3-phosphate dehydrogenase 1 (soluble)
					484	B5381N	D42047	GPD1L	Glycerol-3-phosphate dehydrogenase 1 sample
485	A0961	NM_001482	GATM	Glycine amidinotransferase (L-arginine: glycine amidinotransferase)
					486	B7749	BC023152	GYG2	Glycogen protein 2
487	C0893	BC052210	GARP	Glycoprotein A repeats advantage
					488	A1879	U45955	GPM6B	Glycoprotein M6B
489	C3689	NM_004484	GPC3	Glypican 3
					490	B0738	BQ962682	GOLGA3	The golgi body autoantigen, Golgi apparatus protein subfamily a, 3
491	D8393	AA804745	GBF1	Golgi body specificity brefeldin (brefeldin) A resistance factor 1
					492	A5658	AJ243937	GPSM3	G protein signal conduction modulator 3 (AGS3 sample, Caenorhabditis elegans (C.elegans))
493	C4884	AA036952	Gup1	GRINL1A mixture upstream albumen
					494	C7089	H14263	GAS1	Growth pause specificity 1
495	A6083	NM_004293	GDA	Guanine deaminase
					496	A5969	AA642294	GNG7	Guanine-nucleotide-binding protein (G albumen), gamma 7
497	B8265	AA156792	HEYL	The Hairy/enhancer-of-split sample relevant with the YRPW die body
					498	B3924	AK075151	HSPB7	Heat shock 27kDa protein family, member's 7 (painstaking effort

				Pipe)
					499	A6317	AI205684	HSPA2	Heat shock 70kDa albumen 2
500	F1385	AB002320	NEDL1	HECT contains C2 and WW territory E3 ubiquitin protein ligase 1
					501	C6283	BC039071	HHAT	Hedgehog (Hedgehog) acyltransferase
502	A0451	V00497	HBB	Oxyphorase, beta
					503	C1603	BQ446275	HBD	Oxyphorase, delta
504	B2559	CA426475	HBE1	Oxyphorase, epsilon 1
					505	A0460	X55656	HBG2	Oxyphorase, gamma A
506	A5532	BC063521	HS6ST1	Suleparoid sulfuric acid 6-O-sulfotransferase 1
					507	D0231	AA527435	LOC63928	Hepatocellular carcinoma antigen gene 520
508	B3992	BC078664	HMGA1	The AT-of high mobility family hook (hook) 1
					509	A3112	M60052	HRC	Rich Histidine calcium binding protein
510	C7133	NM_139205	HDAC5	Histone deacetylase 5
					511	B5415	NM_024017	HOXB9	Homology frame B9
512	C6875	AA043381	HOXD10	Homology frame D10
					513	B5353N	BC018099		The people, clone IMAGE:4796019, mRNA
514	D3237	BX109466		The people, clone IMAGE:5276804, mRNA
					515	C3975	BX102181		The people is similar to neural wire albumen (thread protein), clone IMAGE:4102657, mRNA
516	A9187	BC034222	HRLP5	H-rev107 sample albumen 5
					517	A2972	X72475		HRV Fab 027-VL
518	A1379	D49742	HABP2	Hyaluronan conjugated protein 2
					519	A2287	AK127945	HYAL2	Hyaluronoglucosaminidase 2
520	A1807N	BC018986	HPGD	Hydroxy-prostaglandin dehydrogenase 15-(NAD)
					521	A3095	U26726	HSD11B2	Hydroxysteroid (11-beta) dehydrogenase 2
522	C4407	BC050354		Hypothetical gene by the BC014163 support
					523	D8684	BX091957		The LOC387905 that supposes
524	B9722	BQ773658		The LOC402560 that supposes
					525	A8056	XM_496688	LOC152573	Hypothetical protein BC012029
526	D4082	NM_138456	MGC20410	Hypothetical protein BC012330
					527	C7564	BC068590	LOC150356	Hypothetical protein BC012882
528	A6664	H19830	DKFZP434G156	Hypothetical protein DKFZp434G156
					529	A6611	N58556	DKFZp547K111 3	Hypothetical protein DKFZp547K1113
530	C0898	AA319638	DKFZp761L141 7	Hypothetical protein DKFZp761L1417
					531	D6549	BC004888	FLJ10052	Hypothetical protein FLJ10052
532	A6475	BC032508	FLJ10781	Hypothetical protein FLJ10781
					533	B3947	AI075877	FLJ11539	Hypothetical protein FLJ11539
534	C2308	AI281932	FLJ13639	Hypothetical protein FLJ13639
					535	B8081	BM981462	FLJ13710	Hypothetical protein FLJ13710

536	F2724	AK024275	FLJ14213	Hypothetical protein FLJ14213
					537	B4626	BC052574	FLJ20171	Hypothetical protein FLJ20171
538	B4469	AB081837	FLJ20315	Hypothetical protein FLJ20315
					539	A8964	AI091459	FLJ20489	Hypothetical protein FLJ20489
540	D5189	BX101094	FLJ21128	Hypothetical protein FLJ21128
					541	F3313	AK025164	FLJ21511	Hypothetical protein FLJ21511
542	B1006	R34577	FLJ21657	Hypothetical protein FLJ21657
					543	B5905	T83961	FLJ21986	Hypothetical protein FLJ21986
544	B1851	AA032154	FLJ22655	Hypothetical protein FLJ22655
					545	A8061	AA808804	FLJ23469	Hypothetical protein FLJ23469
546	B8353	AY358845	FLJ31166	Hypothetical protein FLJ31166
					547	B6208	AA723586	FLJ31951	Hypothetical protein FLJ31951
548	D4414	AA994364	FLJ32421	Hypothetical protein FLJ32421
					549	B7458N	BC068446	FLJ34658	Hypothetical protein FLJ34658
550	F6944	AL137326	FLJ37478	Hypothetical protein FLJ37478
					551	D5971	AI912733	FLJ40125	Hypothetical protein FLJ40125
552	B4545	AI359551	FLJ90119	Hypothetical protein FLJ90119
					553	A1098	AF000560	LOC126208	Hypothetical protein LOC126208
554	C7585	AK095271	LOC128977	Hypothetical protein LOC128977
					555	C4878	BC040176	LOC130576	Hypothetical protein LOC130576
556	C9677	AL832661	LOC143381	Hypothetical protein LOC143381
					557	D5601	AK056793	LOC145694	Hypothetical protein LOC145694
558	B0277	AA747835	LOC150568	Hypothetical protein LOC150568
					559	D9407	CR749484	LOC152519	Hypothetical protein LOC152519
560	C8269	BC060866	LOC163782	Hypothetical protein LOC163782
					561	C1869	BC046365	LOC253012	Hypothetical protein LOC253012
562	A6532	AY358336	LOC255743	Hypothetical protein LOC255743
					563	B3517	AK097190	LOC285286	Hypothetical protein LOC285286
564	C9503	AA621124	LOC338773	Hypothetical protein LOC338773
					565	C9551	AL133596	LOC339692	Hypothetical protein LOC339692
566	D3168	AA776749	LOC57821	Hypothetical protein LOC57821
					567	D1155	AY358100	MGC11324	Hypothetical protein MGC11324
568	B1230	AA703185	MGC24039	Hypothetical protein MGC24039
					569	D3691	R49126	MGC24039	Hypothetical protein MGC24039
570	B3759	AF067420	MGC27165	Hypothetical protein MGC27165
					571	C1982	AI076840	MGC33926	Hypothetical protein MGC33926
572	B0979	BC030666	MGC33993	Hypothetical protein MGC33993
					573	F0655	AK026196	MGC4172	Hypothetical protein MGC4172
574	B0742	BX648671	MGC61571	Hypothetical protein MGC61571
					575	C0400	BC021290	IMP-2	IGF-II mRNA-conjugated protein 2
576	A3044	BC075840	IGHG1	Immunoglobulin heavy chain constant region gamma 1 (Glm sign)

577	B1676	BC025985	IGHG4	Immunoglobulin heavy chain constant region gamma 4 (G4m sign)
					578	A1032	M87790	IGLC1	Immunoglobulin (Ig) lambda constant region 1 (Mcg sign)
579	A2442	AK074668	ISLR	The rich leucine tumor-necrosis factor glycoproteins that contains immunoglobulin superfamily
					580	C7503	N90724	IGSF4	Immunoglobulin superfamily, the member 4
581	A0560N	NM_000618	IGF1	Type-1 insulin like growth factor (somatomedin C)
					582	A0160N	CR621807	IGFBP1	Insulin-like growth factor binding protein 1
583	A1034	BC004312	IGFBP2	Insulin-like growth factor binding protein 2,36kDa
					584	A4474	AF047433	ITGB4BP	Integrin beta4 is conjugated protein
585	B3893	AY549722	ITLN1	Intelectin 1 (furan type semi-lactosi associativity)
					586	A3829	NM_002182	IL1RAP	The interleukin 1 receptor accessory protein
587	A3037	BC030975	IL1RL1	Interleukin 1 receptor sample 1
					588	B7877	AA056734	IQSEC3	IQ die body and Sec7 territory 3
589	A9295	AY335938	IRX1	Iroquois homology frame albumen 1
					590	F8485	AW452669	IARS	Isoleucine-tRNA synthetic enzyme
591	A2888	BI759204	KLK1	Kallikrein 1, kidney/pancreas/saliva
					592	A3135N	X05332	KLK3	Kallikrein 3, (prostate specific antigen)
593	A3211	M13143	KLKB1	Kallikrein B, blood plasma (the Fletcher factor) 1
					594	A6092	AF208070	KLHL3	Kelch sample 3 (fruit bat)
595	B4062	X14640	KRT13	Keratin sulfate 13
					596	C2083	NM_002277	KRTHA1	Keratin sulfate, hair, acidity, 1
597	D0488	AA405685	UHSKerB	Keratin sulfate, ultra-high-sulfur(UHS), B
					598	A9635	BU621583	KIAA0056	KIAA0056 albumen
599	B6832	BC002378	KIAA0652	The KIAA0652 gene product
					600	A5953	N24235	KIAA0789	The KIAA0789 gene product
601	C9575	XM_032571	KIAA0888	KIAA0888 albumen
					602	A8875	R56087	KIAA0984	KIAA0984 albumen
603	B8035	AL834240	KIAA1576	KIAA1576 albumen
					604	C6986	NM_020946	KIAA1608	KIAA1608
605	B1759	AA629743	KIAA1712	KIAA1712
					606	B8308	NM_001001936	KIAA1914	KIAA1914
607	B4896	AK124139	KIAA1970	KIAA1970 albumen
					608	D6871	AB062483	KIF18A	Kinesin family member 18A
609	D6309	BU608866	KIF5A	Kinesin family member 5A
					610	C8249	NM_000893	KNG1	Prokineticin 1
611	A0005N	NM_153683	KL	Klotho
					612	B6617	NM_153486	LDHD	Serum lactic dehydrogenase D
613	A3061	U07643	LTF	Lactotransferrin
					614	A0227	NM_032737	LMNB2	Lamin B2
615	A0184	NM_000426	LAMA2	Ln, alpha 2 (merosin, amyotrophia congenita disease)

616	A0084	BC075838	LAMB3	Ln, beta 3
					617	C6140	NM_007356	LAMB4	Ln, beta 4
618	B4847	AA490011	LTBP1	Potential transforming growth factor beta conjugated protein 1
					619	A4381N	U81523	leftY2	Left and right sides factor of determination 2
620	F0288	BC080187	LMOD1	Leiomodin 1 (unstriated muscle)
					621	B6844	CR627284	LEPR	Leptin (Leptin) acceptor
622	B6115N	AF097431	LEPRE1	The proteoglycan (leprecan) 1 of rich leucine proline(Pro)
					623	B6180	AF052098	LGI2	Richness leucine tumor-necrosis factor glycoproteins LGI family, the member 2
624	F0967	AB006000	LECT1	The chemokine 1 in white corpuscle cell source
					625	A1779N	AF025534	LILRB5	Leukocytic immunity sphaeroprotein sample acceptor, subfamily B (having TM and ITIM territory), the member 5
626	A6566	AK126536	TSK	Be probably chicken tsukushi directly to homologue
					627	C4755	Z30137	LDB3	The LIM territory is in conjunction with 3
628	A6665	AW450890	LMO3	Only contain LIM territory 3 (rhombotin sample 2)
					629	B3695	BC017312	MGC3047	Limitrin
630	D3586	BC041168	LCN12	NGAL 12
					631	A3736	NM_005577	LPA	Lipoprotein, Lp (a)
632	A8193	AA975742	LRP16	LRP16 albumen
					633	D8515	CR591759	LUM	The chamber proteoglycan
634	C7457	W44613	LY6K	Lymphocyte antigen 6 mixtures, seat K
					635	A0654	BC000458	MAL	Mal, T cytodifferentiation albumen
636	F3821	AL117612	MAL2	Mal, T cytodifferentiation albumen 2
					637	B1723	AA634140	ME2	Malic enzyme 2, NAD (+) dependency, mitochondrial
638	E0706	AW298180	MMP7	Matrix metalloproteinase 7 (matrilysin (matrilysin), the uterus)
					639	C8140	BC006093	MMP9	Matrix metalloproteinase 9 (gelatinase B, 92kDa gelatinase, 92kDa IV Collagen Type VI enzyme)
640	A7972	NM_170677	MEIS2	Meis1, marrow ecotropic virus integration site 1 homologue 2 (mouse)
					641	A6122	AB040529	MAGED4	Melanoma antigen, the D of family, 4
642	F0049	NM_007289	MME	The film Zinc metalloproteinase (neutral endopeptidase, enkephalinase, CALLA, CD10)
					643	A2000	BC014564	MEST	Mesoderm specific transcriptional thing homologue (mouse)
644	C8057	N70019	MT1E	Metallothionein(MT) 1E (functional)
					645	A6757	T95199	MT1F	Metallothionein(MT) 1F (functional)
646	B2396N	N39341	MT1G	Metallothionein(MT) 1G
					647	A2819	NM_005951	MT1H	Metallothionein(MT) 1H
648	A4284	BM458221	MT1X	Metallothionein(MT) 1X
					649	A0450	NM_005953	MT2A	Metallothionein(MT) 2A
650	B9198	AK123132	MSRA	Methionine sulfoxide reductase A

651	C1412	BX648776	LOC253827	Methionine sulfoxide reductase B3
					652	A1423	L38486	MFAP4	Microfibril related protein 4
653	D9341	AI823969	MGST1	Microsome glutathione S-transferase 1
					654	A2162	X91148	MTP	Microsomal triglyceride transfer protein (big polypeptide, 88kDa)
655	D8609	NM_033063	MAP6	Microtubule-associated protein 6
					656	A4705	NM_005928	MFGE8	Dairy fats ball-EGF the factor 8 albumen
657	D0470	BC011873	MTRF1L	Mitochondrial translation releasing hormone 1 sample
					658	A1558N	U66464	MAP4K1	Mitogen activated protein kinase kinase kinase kinases 1
659	B4246	AF131821	MGLL	Monoglyceride lipase
					660	F3349	AL109706		MRNA total length insertion sequence cDNA clones EUROIMAGE 362430
661	F2702	AL049990		MRNA; CDNA DKFZp564G112 (from clone DKFZp564G112)
					662	A3553	J05581	MUC1	Mucoprotein 1, stride film
663	A0720N	L21998	MUC2	Mucoprotein 2, intestines/tracheae
					664	B5338N	AA757932	MINPP1	Multiple inositol polyphosphate Histidine Phosphoric acid esterase, 1
665	C2258	AI093859	MLL5	Marrow/lymph or mixed lineage leukemia 5 (trithorax homologue, fruit bat)
					666	A1886	BC029261	MYOC	Muscle fibrin, trabecular network inducibility glucocorticosteroid is replied
667	D4984	AW977610	MYO1E	Myoglobulin I E
					668	A3489	NM_000260	MYO7A	Myosin VIIA (Usher syndrome 1B (autosomal feminine gender, seriousness))
669	A3296	M24122	MYL3	Myosin, light chain polypeptide 3, alkalescence; Ventricle (ventricular), bone, at a slow speed
					670	A4641	J02854	MYL9	Myosin, light chain polypeptide 9, modulability
671	B2506	AL834231	MTPN	Myotrophin (Myotrophin)
					672	B6305	H03606	NPL	N-sialic acid pyruvic acid lyase (dihydrodipicolinate synthase)
673	B1531	BC063304	NPR1	Natriuratic peptide receptor A/ guanylate cyclase A (atrionatriuretic peptide receptor A)
					674	A1456	M59305	NPR3	Natriuratic peptide receptor C/ guanylate cyclase C (atrionatriuretic peptide receptor C)
675	C7971	AA195950	NRAP	The nebulin anchorin of being correlated with
					676	A4831	D83017	NELL1	NEL sample 1 (chicken)
677	B3699	NM_006617	NES	Neural epidermal stem albumen (nestin)
					678	F1252	AB023193	NTNG1	Lead albumen (netrin) G1
679	A6063	NM_001005388	NFASC	Neurofascin (neurofascin)
					680	A5027	U89165	NRGN	Neural corpuscular protein (neurogranin) (the protein kinase C substrate, RC3)

681	B9126	NM_020795	NLGN2	Nerve is joined albumen (neuroligin) 2
					682	B5787	NM_182964	NAV2	Neurone navigation thing (navigator) 2
683	C9201	NM_000909	NPY1R	Neuropeptide Y Receptors Y1
					684	B0020	NM_001007156	NTRK3	The neurotrophic Tyrosylprotein kinase, acceptor, type 3
685	B0149	AF052090	NNT	Nicotinamide Nucleotide Transhydrogenase
					686	C0472	BQ044958	NRK	The Nik associated kinase
687	C5950	CF146489	NKX3-1	NK3 transcription factor dependency, seat 1 (fruit bat)
					688	A4830	NM_004557	Carve and lack albumen 4	Carve and lack albumen homology thing 4 (fruit bat)
689	B7552	NM_016143	NSFL1C	NSFL1 (p97) cofactor (p47)
					690	A0875	L13740	NR4A1	Nuclear receptor subunit family 4, the A group, the member 1
691	A0805	NM_002505	NFYA	Nuclear factor Y, alpha
					692	B6820	XM_497078	NUP188	Nucleoporin 188kDa
693	C7256	NM_021963	NAP1L2	Nucleosome assembly protein 1 sample 2
					694	B4244	AK128514	NDEL1	NudE nuclear distribution gene E homologue sample 1 (Aspergillus nidulans (A.nidulans))
695	B5969	AF191652	NUDT4	Nudix (nucleoside diphosphate bound fraction X)-pattern body 4
					696	D4979	AW977053	CAS1	The O-Transacetylase
697	F3395	AB032953	ODZ2	Odz, odd number Oz/ten-m homologue 2 (fruit bat)
					698	B4288	AK092766	OLFML3	Smell Jie's albumen sample 3
699	B0997	BQ000754	OR7E47P	Olfactory receptor, family 7, subfamily E, member's 47 pseudogenes
					700	A0472	BG569025	ORM1	Seromucoid (orosomucoid) 1
701	F2316	AB033090	PAK7	P21 (CDKN1A)-activated protein kinase 7
					702	A1275	AF020543	PPT2	Palmityl-protein thioesterase 2
703	A3340	M93284	PNLIPRP2	Pancreas lipase associated protein 2
					704	A6261	L02867	HUMPPA	Secondary tumprigenicity antigen
705	A3085	L04308	PTHR1	Parathyroid hormone acceptor 1
					706	A2970	BM687969	PVALB	Parvalbumin (parvalbumin)
707	A0709N	W20074	PNPLA4	Contain Patatin sample Phospholipid hydrolase territory 4
					708	A0574	NM_033018	PCTK1	PCTAIRE protein kinase 1
709	B7105	AK055782	PDLIM2	PDZ and LIM territory 2 (mystique)
					710	C7651	BM560961	PDLIM3	PDZ and LIM territory 3
711	F3501	AK021708	PDZRN3	Contain pdz domain fourth finger 3
					712	A3242N	BC039733	PTX3	The Pentaxin genes involved is promptly induced by IL-1beta
713	B2142	NM_000277	PAH	Phenylalanine hydroxylase
					714	B7947	NM_080672	PHACTR3	Phosphoric acid esterase and Actin muscle instrumentality 3
715	F5803	AL359590	PGS1	The phosphatidyl phosphoglycerol synthase
					716	A4324	NM_004910	PITPNM1	The phosphatidylinositols transfer protein, film relevant 1
717	D6890	AI003542	PDE11A	Phosphodiesterase 11A
					718	B8389	AK095203	PDE3A	Phosphodiesterase 3A, cGMP-suppresses

719	A3055	AK095384	PDE4C	Phosphodiesterase 4 C, cAMP specificity (phosphodiesterase E1 dunce homologue, fruit bat)
					720	A2307	NM_004563	PCK2	Phosphoenolpyruvate carboxykinase 2 (mitochondrial)
721	A2755	BC011262	PHGDH	Phosphoglycerate dehydrogenase
					722	A1157N	NM_002667	PLN	Phospholamban (phospholamban)
723	A3605	U17033	PLA2R1	Phospholipase A2 acceptor 1,180kDa
					724	B6373	BX423161	LHPP	Phosphoric acid Methionin phosphohistidine inorganic pyrophosphate Phosphoric acid esterase
725	B4239	NM_058179	PSAT1	Phosphoserine aminotransferase 1
					726	A1014	NM_000301	PLG	Plasminogen
727	F0834	NM_000930	PLAT	The plasminogen activator, tissue
					728	B4086	NM_006206	PDGFRA	Thrombocyte source growth factor receptors, the alpha polypeptide
729	D6878	AI002365	PDGFRB	Thrombocyte source growth factor receptors, the beta polypeptide
					730	A6691	NM_021200	PLEKHB1	Contain the pleckstrin homeodomain, the B of family (evectins) member 1
731	A4597	U97519	PODXL	The pedalcalix protein sample
					732	A3208	D29013	POLB	Polysaccharase (at DNA's), beta
733	F6101	AW978378	KCNN3	Potassium intermediate/small-conductance calcium activatory passage, subfamily N, the member 3
					734	A3070N	BC063109	KCNJ1	Potassium inward rectification passage, subfamily J, the member 1
735	A6181	NM_002241	KCNJ10	Potassium inward rectification passage, subfamily J, the member 10
					736	D5416	AF209747	KCNMB2	The big electricity of potassium is led calcium activatory passage, subfamily M, and beta member 2
737	A3447N	M55513	KCNA5	Potassium voltage control passage, the shaker subfamily of being correlated with, the member 5
					738	D3143	AA767342	KCNC3	Potassium voltage control passage, the Shaw subfamily of being correlated with, the member 3
739	B0838	NM_002581	PAPPA	The blood plasma protein A that gestation is relevant, pappalysin 1
					740	C8046	NM_002864	PZP	Pregnoglobulin
741	C8718	AA206141	PRICKLE1	Prickle sample 1 (fruit bat)
					742	D3149	AF338109	PACAP	Apoptosis precursor Caspase adapter (adaptor) albumen
743	F0920	AF098269	PCOLCE2	Precollagen C-endopeptidase reinforce 2
					744	D8901	AI262277	PFN2	Profilin 2
745	A1748	U29089	PRELP	The terminal rich leucine repeat sequence protein of rich proline(Pro) arginine
					746	A3905	AF117225	PROM1	Film conjugated protein (Prominin) 1 that dash forward
747	A3258	U19487	PTGER2	Prostaglin E Receptor 2 (hypotype EP2), 53kDa
					748	B1655	AA455926	PTGER3	Prostaglandin E receptor 3 (hypotype EP3)
749	F0018	NM_000963	PTGS2	Prostaglandin endoperoxide synthase 2 (PGG/H synthase and cyclo-oxygenase)

750	C8119	NM_002775	PRSS11	Proteolytic enzyme, Serine, 11 (IGF associativities)
					751	A3291	BM805032	PRSS2	Proteolytic enzyme, Serine, 2 (trypsinase 2)
752	B2641	BX094063	PIN4	Albumen (peptidyl-prolyl suitable/anti-isomerase) NIMA-interaction, 4 (tiny albumen (parvulin))
					753	A9668	AA759203	XTP7	The albumen 7 of hepatitis B virus X antigen (HBxAg) trans-activation
754	A1816N	BC075800	PRKAR2B	Protein kinase, cAMP dependency, modulability, II type, beta
					755	A8588	BM683764	PRKWNK4	Protein kinase, lysine defect 4
756	D8862	NM_032105	PPP1R12B	Protein phosphatase 1, modulability (inhibitor) subunit 12 B
					757	B7480	AF407165	PPP1R14C	Protein phosphatase 1, modulability (inhibitor) subunit 14 C
758	A2536	U48707	PPP1R1A	Protein phosphatase						1, modulability (inhibitor) subunit 1A
					759	B6647	XM_350880	PPM1H	Protein phosphatase 1H (containing the PP2C territory)
760	A6385	AA663484	PPP2R2B	Protein phosphatase 2 (being 2A originally) is regulated subunit B (PR 52), the beta isotype
					761	C9379	AL049338	PTPRD	Protein-tyrosine-phosphatase, receptor type, D
762	A0860	M55671	PROZ	Albumen Z, the vitamin k-dependent plasma glycoprotein
					763	B2346	AA669023	PCDH9	Former cadherin 9
764	B9131	AA626775	PCDHA6	Former cadherin alpha 6
					765	A2624	NM_002563	P2RY1	Purinergic receptor P2Y, the G albumen coupling, 1
766	A6781	CB529051	G0S2	The lymphocyte G0/G1 switch gene of inferring
					767	A6574N	NM_032932	RAB11FIP4	RAB11 family interaction albumen 4 (II class)
768	B5608N	BC011782	RAB3A	RAB3A, member RAS oncogene family
					769	A1257	BC006992	RAD51AP1	RAD51 related protein 1
770	D5783	BX092660	RORA	The RAR orphan receptor A that is correlated with
					771	B9024	BC042688	RASD1	RAS, induced by dexamethasone 1
772	A6598	BM677885	RASL11B	The RAS sample, family 11, member B
					773	A2202	AJ001016	RAMP3	The albumen 3 of acceptor (thyrocalcitonin) activity modifying
774	B3911	BC038457	DKFZP586H212 3	The mytolin enzyme that regeneration is relevant
					775	A6666	BU728456	RIMS2	Regulate synaptic membrane exocytosis 2
776	A2739	AF073920	RGS6	G protein signal conduction instrumentality 6
					777	B6777	D31888	RCOR1	REST corepressor 1
778	B8016	AA528243	RTN4RL1	Reticulon 4 acceptor samples 1
					779	A3116	M38258	RARG	Retinoic acid receptor (RAR), gamma
780	B9056	AF433662	ARHGEF3	Rho guanine nucleotide exchange factor (GEF) 3
					781	D5408	AA954092	RHBDL2	Rhombohedral (Rhomboid), scun sample 2 (fruit bat)
782	C6829	AB018260	RHOBTB2	Contain the relevant BTB territory 2 of Rho
					783	B2588	H84909	MGC34774	Ribosomal protein L 13A sample

784	B3834	AB033040	RNF150	Ring finger protein 150
					785	D3461	BC028373	RG9MTD2	Contain RNA (the methyltransgerase territory 2 of guanine-9-)
786	B2780	CK822142	RBM5	RNA is in conjunction with die body albumen 5
					787	A7828	NM_017495	RNPC1	Contain RNA-land (RNP1, RRM) 1
788	F2307	AF010236	SGCD	Sarcoglycan (Sarcoglycan), delta (glycoprotein that the 35kDa dystrophin is relevant)
					789	B9057	AF361494	SOSTDC1	Contain Sclerostin territory 1
790	A4424	NM_003966	SEMA5A	The Sema territory, seven thrombospondin tumor-necrosis factor glycoproteinss (1 type and 1 type sample), membrane-spanning domain (TM) and short tenuigenin territory, (brain signal albumen) 5A
					791	F1746	AF389426	SEMA6D	The Sema territory, membrane-spanning domain (TM) and tenuigenin territory, (brain signal albumen) 6D
792	F1976	AB029496	LOC56920	Brain signal albumen sem2
					793	C8190	BX248009	SERPINA4	Serine (or halfcystine) proteinase inhibitor, clade A (alpha-1 protease inhibitor, antitrypsin), the member 4
794	A3024	NM_001756	SERPINA6	Serine (or halfcystine) proteinase inhibitor, clade A (alpha-1 protease inhibitor, antitrypsin), the member 6
					795	D2315	AY358700	SERPINA9	Serine (or halfcystine) proteinase inhibitor, clade A (alpha-1 protease inhibitor, antitrypsin), the member 9
796	A7998	AA521405	SPAP1	The Phosphoric acid esterase anchorin 1 that contains the SH2 territory
					797	F3839	AF131754	SH3BGRL2	The SH3 territory is in conjunction with the albumen sample 2 of rich L-glutamic acid
798	A0917	AF036268	SH3GL2	SH3-territory GRB2 sample 2
					799	B1084	AA682533	SGOL2	Shugoshin sample 2 (schizosaccharomyces pombe)
800	D0657	AB058780	SIAT2	Sialyl transferring enzyme 2 (single sialyl Sphingolipids,sialo sialyl transferring enzyme)
					801	F3433	L13972	SIAT4A	Sialyl transferring enzyme 4A (beta-galactoside alpha-2,3-sialyl transferring enzyme)
802	C8638	BC059363	SIAT7C	Sialyl transferring enzyme 7 ((the neural aminoacyl-2 of alpha-N-acetyl, 3-beta-galactosyl-1,3)-N-acetamino galactosidase alpha-2,6-sialyl transferring enzyme) C
					803	B3997	AY044437	SFXN5	Sideroflexin 5
804	A8433	NM_005843	STAM2	The fit molecule of signal transduction (SH3 territory and ITAM die body) 2
					805	B9455	BQ447358		Be similar to B230208J24Rik albumen
806	D5870	AA972840		Be similar to hypothetical protein
					807	B8401	BU743008		Be similar to the implantation associated protein
808	C1732	AK091965	MGC15937	Be similar to RIKEN cDNA 0610008P16 gene
					809	B4760N	AA721101	LOC92162	Be similar to RIKEN cDNA 2600017H02

810	D6250	BX108635		Be similar to RIKEN cDNA C330003B14
					811	B7600	BC007956	DKFZp727A071	Be similar to tRNA synthetase II class
812	A9462	BM474898	SLIT2	Slit homologue 2 (fruit bat)
					813	A9914	R61832	SNRPB2	Micronuclear ribonucleoprotein polypeptide B "
814	D3947	AI742927	SHPRH	SNF2 histone joint PHD RING helicase
					815	A4875	NM_000336	SCNN1B	The sodium channel, non-voltage-controlled 1, beta (Liddle syndrome)
816	A1533	U38254	SCNN1D	The sodium channel, non-voltage-controlled 1, delta
					817	A4701	U53347	SLC1A5	Solute carrier protein family 1 (neutral amino acid transporter albumen), the member 5
818	A1546	BX647484	SLC12A1	Solute carrier protein family 12 (sodium/potassium/chlorine translocator), the member 1
					819	A3470	NM_003984	SLC13A2	Solute carrier protein family 13 (sodium dependency dicarboxylic acid translocator), the member 2
820	A7358N	NM_022829	SLC13A3	Solute carrier protein family 13 (sodium dependency dicarboxylic acid translocator), the member 3
					821	A6918	BQ183489	SLC14A1	Solute carrier protein family 14 (urea translocator), member 1 (Kidd blood group)
822	A3628	U59299	SLC16A5	Solute carrier protein family 16 (monocarboxylate transporter albumen), the member 5
					823	C0659	AK123243	SLC22A8	Solute carrier protein family 22 (organic anion translocator), the member 8
824	A5748N	AB067483	SLC25A25	Solute carrier protein family 25 (plastosome vehicle; The phosphoric acid vehicle), the member 25
					825	A2189	NM_000112	SLC26A2	Solute carrier protein family 26 (vitriol translocator), the member 2
826	A3911	AF030880	SLC26A4	Solute carrier protein family 26, the member 4
					827	B1020	AF064255	SLC27A5	Solute carrier protein family 27 (fatty acid transport protein), the member 5
828	D6611	AI000771	SLC30A5	Solute carrier protein family 30 (zinc translocator), the member 5
					829	A7155	X77737	SLC4A1	Solute carrier protein family 4, anion-exchange protein, member 1 (erythrocyte membrane protein band 3, Diego blood group)
830	A4675	M95549	SLC5A2	Solute carrier protein family 5 (sodium/glucose cotransporter), the member 2
					831	A8796	AB020532	SLC7A7	Solute carrier protein family 7 (cationic amino acid transporter albumen, y+ system), the member 7
832	B8005	NM_012244	SLC7A8	Solute carrier protein family 7 (cationic amino acid transporter albumen, y+ system), the member 8
					833	D8620	NM_003048	SLC9A2	Solute carrier protein family 9 (sodium/hydrogen exchange albumen), isotype 2
834	C1402	NM_198956	SP8	The Sp8 transcription factor

835	A6689	BU741863	SPOCK	The Sparc/ osteonectin, cwcv and kazal sample territory proteoglycan (testis proteoglycan)
					836	C8088	D87465	SPOCK2	The Sparc/ osteonectin, cwcv and kazal sample territory proteoglycan (testis proteoglycan) 2
837	D6561	AA994999	SYNE2	Contain the spectrin tumor-necrosis factor glycoproteins, nuclear envelope 2
					838	B2953	BG182727	SPDY1	Speedy homologue 1 (fruit bat)
839	A1630N	NM_003117	SPAM1	Sperm adhesion molecule 1 (PH-20 Unidasa, zona pellucida combination)
					840	D3872	AK095036	SPAG16	The antigen 16 that sperm is relevant
841	A6909	NM_018667	SMPD3	Sphingomyelin phosphodiesterase 3, neutral film (neutral sphingomyelinase enzyme II)
					842	D3239	AA112743	SGPL1	Sphingosine-1-phosphate lyase 1
843	F6820	CR749297	SKIP	SPHK1 (Sphingosine kinase type 1) interaction albumen
					844	B7559	AB073386	SGEF	The guanine nucleotide exchange factor that contains Src homology 3 territories
845	D7386	BC071780	UNQ846	SRSR846
					846	A5462	AF389338	SCD4	Stearyl-CoA desaturase 4
847	B7313	AF059203	SOAT2	Sterol O-acyltransferase 2
					848	B8432	AF352729	FLJ12541	The homologue (mouse) that vitamin A acid gene 6 stimulates
849	A0042	AF029082	SFN	Stratifin
					850	D7518	AA007591	SENP5	SUMO1/sentrin specific protease 5
851	D3685	AI911481	SUV420H2	Piebald inhibition 4-20 homologue 2 (fruit bats)
					852	A6670	AB018279	SV2A	Synaptic vesicle glycoprotein 2A
853	A4814	NM_004209	SYNGR3	Cynapse circulating protein 3
					854	B3942	AA191573	SYNJ2	Synaptic vesicle Phosphoric acid esterase 2
855	A5690	AB028952	SYNPO	Cynapse foot albumen
					856	B8366	AI342255	SYNPO2	Cynapse foot albumen 2
857	C6207	AB188489	SYNPO2L	Cynapse foot albumen 2 samples
					858	D3671	AA247642	SNAP23	The associated protein of synaptosome, 23kDa
859	D0385	AK125106	SYTL5	Synaptotagmin sample 5
					860	D3336	AA524150	SDC4	Cohere proteoglycan 4 (amphiglycan, anticoagulant protein glycan)
861	A2780N	NM_003182	TAC1	Tachykinin, precursor 1 (material K, Substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma)
					862	A3465	M74558	SIL	TAL1 (SCL) interrupts the seat
863	B3047	AI361002	TBC1D17	TBC1 territory family, the member 17
					864	A1332	AF054910	TEKT2	Tektin 2 (testis)
865	A1151N	M55618	TNC	Tenascin C (six arms (hexabrachion))
					866	B3377	AA807166	TSGA10	Testes specificity, 10
867	A4895	BC007290	TSPAN-1	Tetratransmembrane 1

868	A2869	AF054839	TSPAN-2	Tetratransmembrane 2
					869	D5273	AA862436	THEA	Thioesterase, fat is correlated with
870	A4717	BC050383	TMPO	Thymopoietin
					871	A4595	U96131	TRIP13	Thyroid Hormone Receptors interaction factor (interactor) 13
872	B4649	BM996064	TJP3	Tight junction protein 3 (closely associating (zonuls occludens) 3)
					873	A1583N	U91963	TLL1	Tolloid sample 1
874	A0461	NM_001068	top2B	Topoisomerase (DNA) II beta 180kDa
					875	B6507	AI929792		The seat of being transcribed
876	C0437	H04828		The seat of being transcribed
					877	C7770	AA165165		The seat of being transcribed
878	B9634	BX110180		The seat of being transcribed
					879	A9694	AA719352		The seat of being transcribed
880	D0852	AA429665		The seat of being transcribed
					881	B9979	CF595488		The seat of being transcribed
882	C6460	W96022		The seat of being transcribed
					883	B7326	Z98489		The seat of being transcribed
884	B5115	T50062		The seat of being transcribed
					885	B1454	AI820955		The seat of being transcribed
886	D5241	AA938971		The seat of being transcribed
					887	A9673	AA609323		The seat of being transcribed
888	C7747	CA314541		The seat of being transcribed
					889	D4261	AA877216		The seat of being transcribed
890	D3102	BX088780		The seat of being transcribed
					891	D5026	AI822035		The seat of being transcribed
892	D7115	BX100620		The seat of being transcribed
					893	D3291	AA766649		The seat of being transcribed
894	D3672	AA809349		The seat of being transcribed
					895	D5414	BX114748		The seat of being transcribed
896	B2956	AA034053		The seat of being transcribed
					897	C4551	N64389		The seat of being transcribed
898	D4230	BF513800		The seat of being transcribed
					899	D3842	AA830448		The seat of being transcribed
900	B3515	AA741001		The seat of being transcribed
					901	B2297	AW197616		The seat of being transcribed
902	B9513	H18456		The seat of being transcribed
					903	B1956	AA663781		The seat of being transcribed
904	B2436	AI220328		The seat of being transcribed
					905	D3092	BX088758		The seat of being transcribed
906	D5402	BX107944		The seat of being transcribed
					907	A7973	BU738725		The seat of being transcribed

908	D5164	AA935766		The seat of being transcribed, moderate is similar to XP_372039.2 and is similar to hypothetical protein (L1H 3 districts)-people [homo sapiens]
					909	C4182	BU681491		The seat of being transcribed, moderate is similar to XP_373053.2 and is similar to microfilament antigen [homo sapiens]
910	B2547	BM725055		The seat of being transcribed, moderate are similar to the hypothetical gene [homo sapiens] that XP_498452.1 is supported by NM_173697
					911	D6981	AW190289		The seat of being transcribed, moderate is similar to XP_517655.1 and is similar to KIAA0825 albumen [chimpanzee (Pan troglodytes)]
912	F7334	AW189299		The seat of being transcribed is similar to NP_062555.1 carboxypeptidase X (M14 family) [homo sapiens] consumingly
					913	D5994	AA975173		The seat of being transcribed is similar to XP_512017.1 hypothetical protein XP_512017 [chimpanzee (Pan troglodytes)] consumingly
914	D5731	AA970389		The seat of being transcribed is similar to XP_529814.1LOC454389[chimpanzee (Pan troglodytes) consumingly]
					915	B2994	BQ185194		The seat of being transcribed faintly is similar to the neural wire albumin A of NP_055301.1 D7c-NTP[homo sapiens]
916	D0634	AA417198		The seat of being transcribed faintly is similar to NP_060190.1 signal transduction adapter (adaptor) albumen-2[homo sapiens]
					917	B1722	AW188090		The seat of being transcribed faintly is similar to NP_080901.1 RIKEN cDNA 1700067P10[house mouse]
918	A6940	AW135188		The seat of being transcribed faintly is similar to XP_509271.1 and is similar to RIKEN cDNA 4921537D05[chimpanzee (Pan troglodytes)]
					919	B1721	AA634326	TCF20	Transcription factor 20 (AR1)
920	A0038N	W73825	TCF21	Transcription factor 21
					921	A7088	AB031046	TCF7L1	Transcription factor 7 samples 1 (T cell-specific, HMG-frame)
922	D2858	AA777755	TFCP2	Transcription factor CP2
					923	F7399	AI928242	TFCP2L1	Transcription factor CP2 sample 1
924	B7571N	BU619137	TGFBR3	Transforming growth factor, and beta receptor II I (the beta glycan, 300kDa)
					925	C4066	NM_013259	TAGLN3	Transgelin 3
926	D6474	NM_001007470	TRPM3	The transient receptor potential cationic channel, subfamily

				M, the member 3
					927	A4792	NM_005723	TM4SF9	Stride film 4 superfamily members 9
928	A8963	N51413	TMAP1	Stride film anchorin 1
					929	C8474	N32157	TMC3	Transmembrane channel sample 3
930	C8660	AA196034	MGC3169	Transmembrane protein 38A
					931	A6233	BM805669	TTR	Transthyretin (prealbumin (prealbumin), amyloid modification I)
932	F6116	BC030244	TNNC1	TnC, at a slow speed
					933	A7154	X74819	TNNT2	TnT 2, heart
934	F0121	AF089854	TU3A	TU3A albumen
					935	B0708	BF673741	TUSC3	Tumor inhibitor candidate 3
936	A7475	D17517	TYRO3	The TYRO3 protein tyrosine kinase
					937	A0930	X51420	TYRP1	Tyrosinase-related protein 1
938	A2978	X04741	UCHL1	Ubiquitin C-terminal esterase L1 (ubiquitin thioesterase (thiolesterase))
					939	B8223	BC041366	USP2	Ubiquitin specific protease 2
940	D5995	AA995921	UPP2	Uridine phosphorylase kinases 2
					941	A3089	AK091961	UMOD	Urine modulation protein (uromucoid, Tamm-Horsfall glycoprotein)
942	A0878	L13288	VIPR1	Vip receptor 1
					943	A0114N	L07868	ERBB4	V-erb-a EBL viral oncogene homologue 4 (birds)
944	A0141	BC002712	MYCN	The relevant oncogene of V-myc myelocytome (myelocytomatosis) virus, (birds) in neuroblastoma source
					945	A0171	NM_002881	RALB	(ras's V-ral ape and monkey leukosis virus oncogene homologue B is correlated with; Gtp binding protein)
946	B0547	AA541526	WDFY3	Contain WD tumor-necrosis factor glycoproteins and FYVE territory 3
					947	C6064	BX647751	WDR20	WD tumor-necrosis factor glycoproteins territory 20
948	F0613	NM_005828	HAN11	The WD-repeat sequence protein
					949	D6252	AA976712	LOC387914	WGAR9166
950	A6672	H27000	WBSCR17	Williams-Beuren syndrome chromosomal region 17
					951	A0342	X51630	WT1	Nephroblastoma (Wilms tumour) 1
952	B0955	AI339506	WAC	Has curling-curling adapter that contains the WW territory (adaptor)
					953	B4291	AK025198	XIST	X (inactivation) specific transcriptional thing
954	D6140	XM_033370	ZFHX2	Zinc refers to homology frame 2
					955	B7727	BC042636	ZNF134	Zinc finger protein 13 4 (clone pHZ-15)
956	A0881N	Z21707	ZNF197	Zinc finger protein 19 7
					957	B6682N	AA180985	FLJ34222	Zinc finger protein 22 9
958	A9121	AB002388	ZNF536	Zinc finger protein 53 6
					959	D3738	AA854756	ZYX	Zyxin
960	B2148	M61900		The plain D synthase of human prostate precursor

961	C0556	H11294		Ym14c11.s1 Soares baby brain 1NIB people cDNA clones IMAGE:480893 '
					962	B3371	AA831240		Oc63h01.s1 NCI_CGAP_GCB1 people cDNA clones IMAGE:13544173 '
963	F2351	AL162042		People mRNA; CDNA DKFZp761L1212 (from clone DKFZp761L1212)
					964	C7105	R50993		Yg63f02.s1 Soares baby brain 1NIB people cDNA clones IMAGE:373733 '
965	E2104	AK000414		People cDNA FLJ20407 fis, clone KAT01658
					966	B0267	R78436		Yi82e09.s1 Soares placenta Nb2HP people cDNA clones IMAGE:1457683 '
967	A9975	AA621665		Af54d01.s1 Soares_ is total _ and fetus _ Nb2HF8_9w people cDNA clone IMAGE:10354573 ' is similar to and contains element MER7 repeat element
					968	D5032	AA931406		Om98c07.s1 NCI_CGAP_Kid3 people cDNA clones IMAGE:15552123 '
969	B1012	AW967446		EST379521 MAGE resequences, MAGJ people cDNA, mRNA sequence
					970	B2883	AA179211		Zp46d09.s1 Stratagene HeLa cell s3 937216 people cDNA clone IMAGE:612497 3 '
971	A6779	AF191687		People's L-Ala-oxoethanoic acid transaminase homologue (TLH6)
					972	D7380	AI016895		Ou31c07.x1 Soares_NFL_T_GBC_S1 people cDNA clones IMAGE:16278843 '

By sxemiquantitative RT-PCR and the selected gene of rna blot analysis checking

In order to prove conclusively the reliability of the data that obtained by the cDNA microarray analysis, to 21 genes (accession number NM_018092, AA632745 of obvious rise are arranged in the information case of suffering from hyaline cell RCC, NM_007250, W86513, BC077726, AA156409, W57613, CR749811, AW972553, AL832896, AK021778, AK026403, AK025204, NM_000677, AF070609, AI290343, AA442590, NM_000552, BC000234, BC034014 and NM_004567) carried out sxemiquantitative RT-PCR experiment.In these genes, by with as the kidney proximal tubule epithelial cell (RPTEC) of normal control relatively, confirmed to have 13 gene (accession number NM_018092, AA632745, NM_007250, W86513, BC077726, AA156409, W57613, CR749811, AW972553, AL832896, AK021778, AK026403 or AK025204) in these four kinds of RCC clones of Caki-1, Caki-2,786-O and A498, raise.Wherein, there are 8 kinds of genes to raise in RCC clone also that (Fig. 2 a).These results are tried microarray analysis in the case with great majority, and height is consistent as a result.Particularly confirmed: compare with RPTEC, the expression amount of C6700 is at ACHN, 769-P, and RXF-63 1L, TUHR10TKB, Caki-1, Caki-2, in these eight kinds of clones of 786-O and A-498 3 kinds raise among A704, OS-RC-2 and the TUHR14TKB (Fig. 2 b).

Graphic for the expression of further investigating these candidate genes, use various cDNA fragments various human tissue and RCC clone to be carried out Northern engram analysis (seeing materials and methods) as probe.In these candidate genes, C6700 (censures and to be that brain signal albumen 5B, the expression of SEMA5B (Genbank accession number BC077726) are limited in the health adult tissue except tire kidney and tire brain (the left drawing of Fig. 3).In addition, utilize the cDNA fragment of C6700 RCC clone to be carried out Northern engram analysis (seeing materials and methods) as probe.(Fig. 3, right drawing) raised in being expressed in of C6700 in 11 kinds of RCC clones three kinds.Then, the USCS database shows, F5749, censure and be ABI gene family member 3 (NESH) conjugated protein (ABI3BP) (Genbank accession number AK025204), be positioned on the karyomit(e) 3q12, and have the transcript of 3545 bases that constitute by 11 exons and the transcript of 4533 bases constituting by 35 exons.Utilize multiple Northern engram analysis that specific probe carries out to show in any health adult tissue, all not have expression by the transcript of 3545 bases that (Fig. 4 a), yet as probe, observe and generally express about 2.0,4.5 and the transcript (Fig. 4 b) of 7.5kb in the health adult tissue with the consensus sequence of 3545 base transcripts and 4533 base transcripts.C8919 censures and is LOC339977, similar (Genbank accession number to hypothetical protein MGC38937; CR749811), be positioned on the 4q11 karyomit(e), and have the transcript of 3348 bases that constitute by 4 exons.Multiple Northern engram analysis shows that the transcript of about 3.4kb only has very faint expression (Fig. 5) in skeletal muscle.B7032N, censure and be PFKFB4 (6-phosphofructokinase-2-kinases/fructose-2,6-diphosphatase 4) gene (GenBank accession number NM_004567), the PFKFB4 transcript of its about 3.5kb is at 2 middle up-regulateds of 11 bladder cancer cell lines, but not expression (Fig. 6) in the normal organ except that testis and pancreas.B9320, censure is that (GenBank accession number: V1 is BC034014 to FBXL16 (F-box and rich leucine repeat sequence protein 16) gene, V2 is NM_153350), two FBXL16 transcripts of its about 4 kb (V1) and 2.4kb (V2) significantly raise in 2 of 4 bladder cancer cell lines.2.4kb transcript has expression in kidney and Tiroidina, yet the transcript of 4kb is not expressed (Fig. 7) in the normal organ except brain, testis, spinal cord and pancreas.

In order further to investigate the characteristic of B7032N, the expression of B7032N and the Subcellular Localization of B7032N gene product in mammalian cell, have been measured.At first, when the expression proteic plasmid of B7032N (pCAGGS-B7032N-HA) transient transfection was in the COS7 cell, the western engram analysis showed that in transfection 24 with after 48 hours, (Figure 10 a) as the big or small band expression of expection for external source B7032N.In addition, immunocytochemical stain has shown that external source all is positioned tenuigenin (Figure 10 b) in all transfectional cells.

In order further to investigate the characteristic of B9320, the expression of B9320 and the Subcellular Localization of B9320 gene product in mammalian cell, have been investigated.At first, will express the proteic plasmid of B9320 (pCAGGS-B9320-HA) transient transfection in the COS7 cell time, the western engram analysis shows, external source B9320 expresses as single band all in transfection 24 with after 48 hours that (Figure 13 a).In addition, immunocytochemical stain is presented at that external source all is positioned tenuigenin (Figure 13 b) in all transfectional cells.

The genome structure of C6700

In order to obtain the global cDNA sequence of C6700, be that template has been carried out RT-PCR with RCC clone OS-RC-2 or A704.C6700 censures and is brain signal albumen 5B, and SEMA5B is positioned on the karyomit(e) 3p21.1.

C6700 has two different variants of being made up of 23 exons of transcribing, and (Fig. 8 a) to correspond respectively to C6700V1 and C6700V2.In the exons 1,16 and 20 of V2, optional variation is arranged, and that other exon is two variants is total, in last exon of V2, produced a new terminator codon, and the terminator codon of V1 is in exon 22.Produced the exons 1 of a new exon of forming by 32bp as the V2 variant.The exons 16 of V2 than the exons 16 of V1 at 3 ' terminal long 3bp, and the extron 20 of V2 also than the extron 20 of V1 at the long 3bp of 5 ' end.In addition, exon 21 is lacked 131bp than the exon 21 of V2 at 3 ' end.The full length cDNA sequence of C6700V1 and C6700V2 variant is made of 4725 and 4494 Nucleotide respectively.The ORF of these variants begins in exons 1 separately.Finally, encode respectively 1093 and 1152 amino acid of V1 and V2 transcript.In order to confirm that further the expression of each variant in RCC clone is graphic, utilize the primer sets of every kind of variant of identification to carry out sxemiquantitative RT-PCR (seeing Fig. 8 b).As a result, found that the V2 variant crosses high expression level (Fig. 8 b) specifically in the RCC cell.Therefore, carried out further functional analysis for C6700V2.

Be designed for the growth of the siRNA (siRNA) that reduces C6700, B7032N and B9320 expression Retarding effect

In order to assess the growth promoting function of C6700, B7032N and B9320, the expression of endogenous C6700, B7032N and B9320 in having struck RCC clone low by RNA perturbation technique based on the mammalian cell carrier, wherein, for C6700, use OS-RC-2 clone, it comprises and shows that C6700 crosses the cell of high expression level; For B7032N, use to show that B7032N crosses the RXF-631L and the A498 of high expression level; For B9320, use to show that B9320 crosses the Caki-2 and the A498 (seeing materials and methods) of high expression level.The expression level of C6700, B7032N and B9320 is investigated by sxemiquantitative RT-PCR experiment.

Shown in Fig. 9 a, compare with four kinds of contrast siRNA constructs (psiU6BX-luciferase, out of order, EGFP and simulation), the special siRNA (si1, si6 and si-#2) of C6700 has suppressed the expression of C6700.Compare with contrast siRNA (si-out of order and si-simulation), in these siRNA constructs of detection, si-#2 has reduced the expression of SEMA5B mRNA effectively, and (Fig. 9 a).The obvious decline (Fig. 9 c) of colony number of cell and viable count purpose that MTT records obviously descend (Fig. 9 b) have been observed in the cell after utilizing si-#2 to handle.In order to get rid of the miss the target possibility of effect of SEMA5B-siRNA (si-#2), prepared siRNA with two kinds of forms of 3-bp alternate.Found that: in the OS-RC-2 cell, these two kinds of siRNA do not suppress the expression (data not shown) of SEMA5B.These results suggest C6700 has significant function in the cell survival of RCC and/or growth.

As shown in figure 11, with the contrast siRNA construct (si-is out of order) compare, B7032N (si#4) suppressed this expression of gene (Figure 11 a, 11b).Utilize colony forming assay that these siRNA constructs carry out and MTT to measure and show, import PFKFB4-si#4 and suppressed RXF-631L (Figure 11 a) and the growth of A498 (Figure 11 b) cell.In order further to confirm the growth enhancing effect of B7032N, set up stable expression of exogenous B7032N the NIH3T3 derived cell (B7032N-A ,-B ,-C and-the D cell).The Western engram analysis shows: (Figure 12 a) to have very high external source B7032N protein level in four are derived the clone.Subsequently MTT measures and has shown that four kinds of derived cells systems are B7032N-A ,-B ,-C and-the D cell, than transfection the cell of simulation plasmid (simulation-A ,-B and-the C cell) growth faster (Figure 12 b), the expression that shows B7032N may strengthen cell and grow.These results hint that B7032N has carcinogenic (oncogenic) effect to RCC.

As shown in figure 14, compare with contrast siRNA construct (si-is out of order), B9320 (si#2) has suppressed this expression of gene (Figure 14) significantly.Utilizing colony forming assay that these siRNA constructs carry out and MTT to measure shows: import the specific siRNA of FBXL16 and suppressed Caki-2 (Figure 14 a) and the growth of A498 (Figure 14 b) cell.In order further to confirm the growth enhancing effect of B9320, set up the NIH3T3 derived cell (FBXL16-1 ,-2 ,-3 and-4 cells) of stable expression of exogenous B9320.The Western engram analysis shows: (Figure 15 a) to have very high external source B9320 protein level in four are derived the clone.Subsequently MTT measures and shows that four kinds of derived cells systems are FBXL16-1 ,-2 ,-3 and-4 cells, than transfection cell (simulation-1 ,-2 and-3 cells) growth faster (Figure 15 b) of simulation plasmid, show that the expression of B9320 may be able to strengthen the cell growth.These results hint that B9320 has carcinogenic (oncogenic) effect to RCC.

Table 6. is at C6700, the oligonucleotide sequence of B7032N and 9320 siRNA

Discuss

Our understanding for the various human tumour origin cause of formation has been promoted in nearest molecular biological progress.For RCC, several study group have reported molecular pattern analysis (Yao M, et al., (2005) the J Pathol. based on microarray; 205:377-87; Liou LS, et al., (2004) BMC Urol.; 4:9; TakahashiM, et al., (2001) Proc Natl Acad Sci U S A.; 98:9754-9; Boer JM, et al., (2001) Genome Res.; 11:1861-70; Young AN, et al., (2001) Am J Pathol.; 158:1639-51; Higgins JP, et al., (2003) Am J Pathol.; 162:925-32; Skubitz KM and Skubitz AP. (2002) J Lab Clin Med.; 140:52-64.).Though having highlighted some, these researchs may be used for the candidate gene of diagnostic markers, but because the tumor mass mixture of various kinds of cell colony normally, except cancer cells, also comprise for example inflammatory cell, stroma cell and inoblast etc., and the shared ratio of every kind of cell type has clearly variation with the difference of individuality, so, may not reflect variation in the kidney generating process definitely based on the data of isolating mRNA from tumor mass.In addition, RCC is considered to derive from the kidney proximal tubule epithelial cell in the renal cortex.Therefore, previously disclosed microarray data may be subjected to contrasting the remarkably influenced of cell heterogeneity in the prepared product.In the present invention, carry out the pure colony that laser microbeam micro-dissection (LMM) obtains kidney cancer cell and contrasts normal renal cortical cell.With a kind of representative transparent cell RCC (ccRCC) expression pattern data (Takahashi M, et al., (2003) Oncogene. of utilizing the RNA that from overall (bulk) tumor tissues, obtains to obtain by microarray; 22:6810-8.) compare, in 85 genes (table 4) that in surpassing 75% ccRCC information case of the present invention, generally raise, 4 (5%) and overall expression pattern data overlaid (Takahashi M, et al., (2003) Oncogene. are only arranged; 22:6810-8.).These differences are because the RCC cell is allogenic on histopathology, may be subjected to obvious influence owing to sneaking into of non-cancerous cells from the expression pattern of overall tissue.Surprisingly, in our list of genes (table 4), raise the most tangible preceding 24 genes all be not included in before from the result of the ccRCC expression pattern of overall tissue acquisition (Yao M, et al., (2005) J Pathol.; 205:377-87; Takahashi M, et al., (2003) Oncogene.; 22:6810-8.).This evidence prompting, microarray of the present invention is more reliable than the data of other report.This difference may be because kidney medulla is sneaked into in the normal kidney tissue that compares, and has obviously disturbed the expression pattern from the general cell acquisition before this.For example, high expression level in renocortical kidney proximal tubule epithelial cell but not in kidney medulla the gene of high expression level, may selected conduct totally organize downward modulation or unconverted gene in the expression pattern.Consider above these factors, from the surgical operation sample, utilize the LMM system as much as possible carcinous and colony normal epithelium cell of purifying be very important.

Expressing the gene that changes in most of ccRCC can perhaps may be the reason that kidney takes place as the target of diagnosis molecular marker and treatment RCC.In the gene that raises, NNMT (NNMT), IGFBP3 (insulin-like growth factor binding protein 3), ENPP3 (the outer Nucleotide Pyrophosphate phosphohydrolase/phosphodiesterase 3 of born of the same parents), VEGF (vascular endothelial growth factor) and VWF (the VonWillebrand factor) and other comprise in this application.Having reported NNMT and having compared in the RCC of other type in ccRCC frequently increases, and it is associated with the good prognosis of RCC (Yao M, et al., (2005) J Pathol.; 205:377-87.).Immunohistochemical staining shows that IGFBP-3 causes the imbalance of IGF axle (Takahashi M, et al., (2005) the Int JOncol. among the ccRCC; 26:923-31; Cheung CW, et al., (2004) Kidney Int.; 65:1272-9.).Prove that also IGFP-3 plays some effects (TakahashiM, et al., (2005) Int J Oncol. in the mode of autocrine in the adjusting of RCC cell proliferation; 26:923-31; Cheung CW, et al., (2004) KidneyInt.; 65:1272-9.).Born of the same parents are Nucleotide Pyrophosphate phosphohydrolase (ecto-nucleotide pyrophosphatase)/phosphodiesterase-I enzyme (E-NPP) outward, shears the phosphodiester bond and the phosphoric acid thioester bond of multiple substrate.Mammiferous E-NPP is a protein family that is made of three kinds of albumen that is closely related: E-NPP1, E-NPP2 and E-NPP3.The function of ENPP3 is unknown at present, but the existing ENPP3 that reports that the expression ratio of ENPP3 in tumor tissues is high in the tumour surrounding tissue, detected special form in cholangiocarcinoma (BDC) patient's serum, and ENPP3 promotes cell migration.Therefore, ENPP3 may be relevant with the wetting property of tumprigenicity BDC, and can be used as tumor-marker (Yano Y, et al., (2004) CancerLett.; 207:139-47.).The level of VEGF and vWF is considered to the predictability sign of immunomodulator in the serum, and such immunomodulator comprises interleukin-22 and IFN-alpha; Compare with the patient with low serum level VEGF and vWF, these the two kinds of proteic patients with higher level are for these therapeutic responses not good (Braybrooke JP, et al., (2000) Clin Cancer Res.; 6:4697-704.).In addition, ABCG1 (ATP is in conjunction with box, subfamily G (WHITE), the member 1) and STC2 (bony fish calsequestrin 2) ABCG1, mediate the transhipment of cholesterol usually.People such as Hernan have reported by microarray analysis and difference demonstration and have learnt that ABCG1 raises (Yano Y, et al., (2004) Cancer Lett. in the neck squamous cell carcinoma; 207:139-47.).

On the other hand, in the gene of downward modulation, WT1 (wilms' tumor 1), CDKN1C (cyclin rely on kinase inhibitor 1C) and GAS1 (cessation of growth cessation specificity 1) have shown their effect (Niu Z in growth-inhibiting or apoptosis, et al., (2005) J Urol.; 174:1460-2; Kikuchi T, et al., (2002) Oncogene.; 21:274 1-9; Watanabe H, et al., (1 998) Proc Natl AcadSci USA.; 95:1392-7; Evdokiou A ﹠amp; Cowled PA. (1998) Int JCancer.; 75:568-77).People such as Niu Z. have reported with corresponding normal kidney tissue and have compared that (Niu Z, et al., (2005) J Urol. are obviously reduced in being expressed in of WT1 in the RCC tumour; 174:1460-2.), between the clinical pathological characteristic of RCC and its mRNA level tangible dependency is not arranged although point out.Known CDKN1C can come the inducing cell cycle arrest by the activity that suppresses cell cycle protein dependent kinase.Reported this gene at for example colorectal carcinoma, cancer of the stomach, the epigenetic silence in the noumenal tumours such as hepatocellular carcinoma and carcinoma of the pancreas (Epigenetic silencing) (Kikuchi T, et al., (2002) Oncogene.; 21:2741-9.), prompting CDKN1C works as tumor suppressor gene.Also obviously downward modulation in 14 of 15 cases of the present invention of GAS1.Reported and induced GAS1 in neuronal cell, to suppress the propagation of cell and/or cause apoptosis, and the expression of GAS1 neuroglial propagation (the Evdokiou A ﹠amp that slows down; Cowled PA. (1998) Int J Cancer; 75:568-77.).

In our up-regulated gene of tabulation, special concern brain signal albumen 5B of the present invention (Semaphorin 5B) is (SEMA5B) as the possible molecular target of RCC treatment because it in RCC trans continually activation and in any health adult tissue that the contriver investigated its expression level all detect less than.In the context of the present invention, proved and by siRNA its expression level has been struck lowly, significantly suppressed the growth of RCC cell specifically, shown its vital role in promoting the cell growth.SEMA5B is a cell surface and secretion glycoprotein family, and these albumen belong to gene (Kolodkin AL, et al., (1993) Cell. of the diversified coding growth targeting signal of a group (growth guidance cues); 75:1389-99.).Brain signal albumen and their acceptor, clump albumen (plexins) is identified at first in neural system, needs them to set up correct neural network in neural system.Their scope has expanded to the growth that several non-neural system processes comprise heart and bone, and immunne response and epithelium form take place.Recently, indicated effect (the Tamagnone L ﹠amp of brain signal albumen in tumor growth and transfer; Comoglio PM. (2004) EMBO Rep.; 5:356-61.).In this manual, shown that SEMA5B frequently expresses in clear cell type RCC and RCC clone, meaned that with SEMA5B may be a kind of promising approach of the new medicine of exploitation as target.

In this report,, identified B7032N and the B9320 remarkable high expression level of crossing in the RCC cell by the accurate expression pattern of (though) bladder cancer.The Northern engram analysis that carries out for multiple health adult tissue and RCC clone shows: the expression of B7032N in the health adult tissue except testis and pancreas almost detect less than, and in inspected normal people's vital tissues, almost detect expression less than B9320.This conclusion shows: these genes can become the valuable target of exploitation at the carcinostatic agent of RCC.In addition, the present invention shows: the inhibition that has caused cell growth in the RCC cell is hanged down in striking of endogenous B7032N and B9320.In a word, reported that B7032N and B9320 demonstrate growth-promoting activity in mammalian cell, and the low growth that has suppressed transitional cell bladder carcinoma cell line has been struck in its expression by siRNA.The medicine of exploitation antagonism B7032N and B9320 function may be a kind of rational strategy of cancer therapy.Though the further analysis to B7032N and B9320 function is necessary, the data that proposed should be contributed to some extent to the understanding of deepening the RCC oncogenesis and the new therapy of developing RCC.

On the whole, the up-regulated gene of identifying by accurate RCC expression pattern of the present invention can make us that kidney is had better understanding, and RCC treats and recruit's target of diagnosing tumor sign provides useful information for development is used for.

Industrial applicibility

The renal cell carcinoma gene expression analysis that the coupling laser capture is dissected and full genome cDNA microarray obtains that passes through that this specification sheets is put down in writing has been identified as the specific gene of cancer prevention with the treatment target.Based on the expression of the subclass of the gene of these differential expressions, the present invention provides the molecular diagnosis sign for identifying or detecting renal cell carcinoma.

The method of this specification sheets record also can be used for identifying other molecular target of prevention, diagnosis and treatment renal cell carcinoma.The data of this specification sheets report have increased the overall understanding for renal cell carcinoma, have promoted the exploitation of new diagnosis policy, and the molecular target that is used for the treatment of medicine or prophylactic agent for evaluation provides clue.This information has been made contribution to the more deep formation of understanding renal cell carcinoma, and for developing diagnosis, treatment and finally preventing the New Policy of renal cell carcinoma that enlightenment is provided.

Shown in this specification sheets, can cell growth inhibiting by the siRNA (siRNA) of selectively targeted gene C 6700, B7032N or B9320 gene.Therefore, new siRNA is the useful target of exploitation cancer therapy drug.For example, check C6700, B7032N or B9320 expression or suppress its active material can be used as for example carcinostatic agent of renal cell carcinoma of carcinostatic agent, particularly conduct treatment kidney, be used for the treatment of purposes.

All patents, patent application and the publication that this specification sheets is mentioned incorporated this specification sheets into as a reference all in full.In addition, although at length and with reference to specific embodiments of the present invention described the present invention, will be understood that above-mentioned specification sheets is actually exemplary and indicative, is intended to illustrate the present invention and preferred embodiment thereof.By the experiment of routine, it will be readily appreciated by those skilled in the art that and to carry out various variations and modification to the present invention and do not deviate from the spirit and scope of the present invention.Therefore, the present invention also is not intended to be subjected to the qualification of above-mentioned explanation, and is limited by following claim and equivalent thereof.

Sequence table

＜110〉Oncotherapy Science Inc (ONCOTHERAPY SCIENCE, INC.)

＜120〉method of diagnosis and treatment renal cell carcinoma

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<150>US 60/703,640

<151>2005-07-28

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＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>54

aaaacgtacg cggaatactt cgatctcttg aatcgaagta ttccgcgtac g 51

<210>55

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉S iRNA target sequence

<400>55

cgtacgcgga atacttcga 19

<210>56

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉S iRNA hairpin structure

<400>56

cgtacgcgga atacttcgat tcaagagatc gaagtattcc gcgtacg 47

<210>57

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>57

caccgcgcgc tttgtaggat tcgttcaaga gacgaatcct acaaagcgcg c 51

<210>58

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>58

aaaagcgcgc tttgtaggat tcgtctcttg aacgaatcct acaaagcgcg c 51

<210>59

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA target sequence

<400>59

gcgcgctttg taggattcg 19

<210>60

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA hairpin structure

<400>60

gcgcgctttg taggattcgt tcaagagacg aatcctacaa agcgcgc 47

<210>61

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>61

caccgtgtct tcaagcttga agactatttt 30

<210>62

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>62

aaaatagtct tcaagcttga agacacggtg 30

<210>63

<211>4725

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(198)..(3476)

<400>63

caattcggcc tcgctccttg tgattgcgct aaaccttccg tcctcagctg agaacgctcc 60

accacctccc cggatcgctc atctcttggc tgccctccca ctgttcctga tgttatttta 120

ctccccgtat cccctactcg ttcttcacaa ttctgtaggt gagtggttcc agctggtgcc 180

tggcctgtgt ctcttgg atg ccc tgt ggc ttc agt ccg tct cct gtt gcc 230

Met Pro Cys Gly Phe Ser Pro Ser Pro Val Ala

1 5 10

cac cac ctc gtc cct ggg ccg cct gat acc cca gcc caa cag cta agg 278

His His Leu Val Pro Gly Pro Pro Asp Thr Pro Ala Gln Gln Leu Arg

15 20 25

tgt gga tgg aca gta ggg ggc tgg ctt ctc tca ctg gtc agg ggt ctt 326

Cys Gly Trp Thr Val Gly Gly Trp Leu Leu Ser Leu Val Arg Gly Leu

30 35 40

ctc ccc tgt ctg cct ccc gga gct agg act gca gag ggg cct atc atg 374

Leu Pro Cys Leu Pro Pro Gly Ala Arg Thr Ala Glu Gly Pro Ile Met

45 50 55

gtg ctt gca ggc ccc ctg gct gtc tcg ctg ttg ctg ccc agc ctc aca 422

Val Leu Ala Gly Pro Leu Ala Val Ser Leu Leu Leu Pro Ser Leu Thr

60 65 70 75

ctg ctg gtg tcc cac ctc tcc agc tcc cag gat gtc tcc agt gag ccc 470

Leu Leu Val Ser His Leu Ser Ser Ser Gln Asp Val Ser Ser Glu Pro

80 85 90

agc agt gag cag cag ctg tgc gcc ctt agc aag cac ccc acc gtg gcc 518

Ser Ser Glu Gln Gln Leu Cys Ala Leu Ser Lys His Pro Thr Val Ala

95 100 105

ttt gaa gac ctg cag ccg tgg gtc tct aac ttc acc tac cct gga gcc 566

Phe Glu Asp Leu G1n Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly Ala

110 115 120

cgg gat ttc tcc cag ctg gct ttg gac ccc tcc ggg aac cag ctc atc 614

Arg Asp Phe Ser Gln Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu Ile

125 130 135

gtg gga gcc agg aac tac ctc ttc aga ctc agc ctt gcc aat gtc tct 662

Val Gly Ala Arg Asn Tyr Leu Phe Arg Leu Ser Leu Ala Asn Val Ser

140 145 150 155

ctt ctt cag gcc aca gag tgg gcc tcc agt gag gac acg cgc cgc tcc 710

Leu Leu Gln Ala Thr Glu Trp Ala Ser Ser Glu Asp Thr Arg Arg Ser

160 165 170

tgc caa agc aaa ggg aag act gag gag gag tgt cag aac tac gtg cga 758

Cys Gln Ser Lys Gly Lys Thr Glu Glu Glu Cys Gln Asn Tyr Val Arg

175 180 185

gtc ctg atc gtc gcc ggc cgg aag gtg ttc atg tgt gga acc aat gcc 806

Val Leu Ile Val Ala Gly Arg Lys Val Phe Met Cys Gly Thr Asn Ala

190 195 200

ttt tcc ccc atg tgc acc agc aga cag gtg ggg aac ctc agc cgg act 854

Phe Ser Pro Met Cys Thr Ser Arg Gln Val Gly Asn Leu Ser Arg Thr

205 210 215

att gag aag atc aat ggt gtg gcc cgc tgc ccc tat gac cca cgc cac 902

Ile Glu Lys Ile Asn Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg His

220 225 230 235

aac tcc aca gct gtc atc tcc tcc cag ggg gag ctc tat gca gcc acg 950

Asn Ser Thr Ala Val Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala Thr

240 245 250

gtc atc gac ttc tca ggt cgg gac cct gcc atc tac cgc agc ctg ggc 998

Val Ile Asp Phe Ser Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu Gly

255 260 265

agt ggg cca ccg ctt cgc act gcc caa tat aac tcc aag tgg ctt aat 1046

Ser Gly Pro Pro Leu Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu Asn

270 275 280

gag cca aac ttc gtg gca gcc tat gat att ggg ctg ttt gca tac ttc 1094

Glu Pro Asn Phe Val Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe

285 290 295

ttc ctg cgg gag aac gca gtg gag cac gac tgt gga cgc acc gtg tac 1142

Phe Leu Arg Glu Asn Ala Val Glu His Asp Cys Gly Arg Thr Val Tyr

300 305 310 315

tct cgc gtg gcc cgc gtg tgc aag aat gac gtg ggg ggc cga ttc ctg 1190

Ser Arg Val Ala Arg Val Cys Lys Asn Asp Val Gly Gly Arg Phe Leu

320 325 330

ctg gag gac aca tgg acc aca ttc atg aag gcc cgg ctc aac tgc tcc 1238

Leu Glu Asp Thr Trp Thr Thr Phe Met Lys Ala Arg Leu Asn Cys Ser

335 340 345

cgc ccg ggc gag gtc ccc ttc tac tat aac gag ctg cag agt gcc ttc 1286

Arg Pro Gly Glu Val Pro Phe Tyr Tyr Asn Glu Leu Gln Ser Ala Phe

350 355 360

cac ttg ccg gag cag gac ctc atc tat gga gtt ttc aca acc aac gta 1334

His Leu Pro Glu Gln Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn Val

365 370 375

aac agc atc gcg gct tct gct gtc tgc gcc ttc aac ctc agt gct atc 1382

Asn Ser Ile Ala Ala Ser Ala Val Cys Ala Phe Asn Leu Ser Ala Ile

380 385 390 395

tcc cag gct ttc aat ggc cca ttt cgc tac cag gag aac ccc agg gct 1430

Ser Gln Ala Phe Asn Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg Ala

400 405 410

gcc tgg ctc ccc ata gcc aac ccc atc ccc aat ttc cag tgt ggc acc 1478

Ala Trp Leu Pro Ile Ala Asn Pro Ile Pro Asn Phe Gln Cys Gly Thr

415 420 425

ctg cct gag acc ggt ccc aac gag aac ctg acg gag cgc agc ctg cag 1526

Leu Pro Glu Thr Gly Pro Asn Glu Asn Leu Thr Glu Arg Ser Leu Gln

430 435 440

gac gcg cag cgc ctc ttc ctg atg agc gag gcc gtg cag ccg gtg aca 1574

Asp Ala Gln Arg Leu Phe Leu Met Ser Glu Ala Val Gln Pro Val Thr

445 450 455

ccc gag ccc tgt gtc acc cag gac agc gtg cgc ttc tca cac ctc gtg 1622

Pro Glu Pro Cys Val Thr Gln Asp Ser Val Arg Phe Ser His Leu Val

460 465 470 475

gtg gac ctg gtg cag gct aaa gac acg ctc tac cat gta ctc tac att 1670

Val Asp Leu Val Gln Ala Lys Asp Thr Leu Tyr His Val Leu Tyr Ile

480 485 490

ggc acc gag tcg ggc acc atc ctg aag gcg ctg tcc acg gcg agc cgc 1718

Gly Thr Glu Ser Gly Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser Arg

495 500 505

agc ctc cac ggc tgc tac ctg gag gag ctg cac gtg ctg ccc ccc ggg 1766

Ser Leu His Gly Cys Tyr Leu Glu Glu Leu His Val Leu Pro Pro Gly

510 515 520

cgc cgc gag ccc ctg cgc agc ctg cgc atc ctg cac agc gcc cgc gcg 1814

Arg Arg Glu Pro Leu Arg Ser Leu Arg Ile Leu His Ser Ala Arg Ala

525 530 535

ctc ttc gtg ggg ctg aga gac ggc gtc ctg cgg gtc cca ctg gag agg 1862

Leu Phe Val Gly Leu Arg Asp Gly Val Leu Arg Val Pro Leu Glu Arg

540 545 550 555

tgc gcc gcc tac cgc agc cag ggg gca tgc ctg ggg gcc cgg gac ccg 1910

Cys Ala Ala Tyr Arg Ser Gln Gly Ala Cys Leu Gly Ala Arg Asp Pro

560 565 570

tac tgt ggc tgg gac ggg aag cag caa cgt tgc agc aca ctc gag gac 1958

Tyr Cys Gly Trp Asp Gly Lys Gln Gln Arg Cys Ser Thr Leu Glu Asp

575 580 585

agc tcc aac atg agc ctc tgg acc cag aac atc acc gcc tgt cct gtg 2006

Ser Ser Asn Met Ser Leu Trp Thr Gln Asn Ile Thr Ala Cys Pro Val

590 595 600

cgg aat gtg aca cgg gat ggg ggc ttc ggc cca tgg tca cca tgg caa 2054

Arg Asn Val Thr Arg Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp Gln

605 610 615

cca tgt gag cac ttg gat ggg gac aac tca ggc tct tgc ctg tgt cga 2102

Pro Cys Glu His Leu Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys Arg

620 625 630 635

gct cga tcc tgt gat tcc cct cga ccc cgc tgt ggg ggc ctt gac tgc 2150

Ala Arg Ser Cys Asp Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp Cys

640 645 650

ctg ggg cca gcc atc cac atc gcc aac tgc tcc agg aat ggg gcg tgg 2198

Leu Gly Pro Ala Ile His Ile Ala Asn Cys Ser Arg Asn Gly Ala Trp

655 660 665

acc ccg tgg tca tcg tgg gcg ctg tgc agc acg tcc tgt ggc atc ggc 2246

Thr Pro Trp Ser Ser Trp Ala Leu Cys Ser Thr Ser Cys Gly Ile Gly

670 675 680

ttc cag gtc cgc cag cga agt tgc agc aac cct gct ccc cgc cac ggg 2294

Phe Gln Val Arg Gln Arg Ser Cys Ser Asn Pro Ala Pro Arg His Gly

685 690 695

ggc cgc atc ttc gtg ggc aag agc cgg gag gaa cgg ttc tgt aat gag 2342

Gly Arg Ile Phe Val Gly Lys Ser Arg Glu Glu Arg Phe Cys Asn Glu

700 705 710 715

aac acg cct tgc ccg gtg ccc atc ttc tgg gct tcc tgg ggc tcc tgg 2390

Asn Thr Pro Cys Pro Val Pro Ile Phe Trp Ala Ser Trp Gly Ser Trp

720 725 730

agc aag tgc agc agc aac tgt gga ggg ggc atg cag tcg cgg cgt cgg 2438

Ser Lys Cys Ser Ser Asn Cys Gly Gly Gly Met Gln Ser Arg Arg Arg

735 740 745

gcc tgc gag aac ggc aac tcc tgc ctg ggc tgc ggc gag ttc aag acg 2486

Ala Cys Glu Asn Gly Asn Ser Cys Leu Gly Cys Gly Glu Phe Lys Thr

750 755 760

tgc aac ccc gag ggc tgc ccc gaa gtg cgg cgc aac acc ccc tgg acg 2534

Cys Asn Pro Glu Gly Cys Pro Glu Val Arg Arg Asn Thr Pro Trp Thr

765 770 775

ccg tgg ctg ccc gtg aac gtg acg cag ggc ggg gca cgg cag gag cag 2582

Pro Trp Leu Pro Val Asn Val Thr Gln Gly Gly Ala Arg Gln Glu Gln

780 785 790 795

cgg ttc cgc ttc acc tgc cgc gcg ccc ctt gca gac ccg cac ggc ctg 2630

Arg Phe Arg Phe Thr Cys Arg Ala Pro Leu Ala Asp Pro His Gly Leu

800 805 810

cag ttc ggc agg aga agg acc gag acg agg acc tgt ccc gcg gac ggc 2678

Gln Phe Gly Arg Arg Arg Thr Glu Thr Arg Thr Cys Pro Ala Asp Gly

815 820 825

tcc ggc tcc tgc gac acc gac gcc ctg gtg gag gtc ctc ctg cgc agc 2726

Ser Gly Ser Cys Asp Thr Asp Ala Leu Val Glu Val Leu Leu Arg Ser

830 835 840

ggg agc acc tcc ccg cac acg gtg agc ggg ggc tgg gcc gcc tgg ggc 2774

Gly Ser Thr Ser Pro His Thr Val Ser Gly Gly Trp Ala Ala Trp Gly

845 850 855

ccg tgg tcg tcc tgc tcc cgg gac tgc gag ctg ggc ttc cgc gtc cgc 2822

Pro Trp Ser Ser Cys Ser Arg Asp Cys Glu Leu Gly Phe Arg Val Arg

860 865 870 875

aag aga acg tgc act aac ccg gag ccc cgc aac ggg ggc ctg ccc tgc 2870

Lys Arg Thr Cys Thr Asn Pro Glu Pro Arg Asn Gly Gly Leu Pro Cys

880 885 890

gtg ggc gat gct gcc gag tac cag gac tgc aac ccc cag gct tgc cca 2918

Val Gly Asp Ala Ala Glu Tyr Gln Asp Cys Asn Pro Gln Ala Cys Pro

895 900 905

gtt cgg ggt gct tgg tcc tgc tgg acc tca tgg tct cca tgc tca gct 2966

Val Arg Gly Ala Trp Ser Cys Trp Thr Ser Trp Ser Pro Cys Ser Ala

910 915 920

tcc tgt ggt ggg ggt cac tat caa cgc acc cgt tcc tgc acc agc ccc 3014

Ser Cys Gly Gly Gly His Tyr Gln Arg Thr Arg Ser Cys Thr Ser Pro

925 930 935

gca ccc tcc cca ggt gag gac atc tgt ctc ggg ctg cac acg gag gag 3062

Ala Pro Ser Pro Gly Glu Asp Ile Cys Leu Gly Leu His Thr Glu Glu

940 945 950 955

gca cta tgt gcc aca cag gcc tgc cca ggc tgg tcg ccc tgg tct gag 3110

Ala Leu Cys Ala Thr Gln Ala Cys Pro Gly Trp Ser Pro Trp Ser Glu

960 965 970

tgg agt aag tgc act gac gac gga gcc cag agc cga agc cgg cac tgt 3158

Trp Ser Lys Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser Arg His Cys

975 980 985

gag gag ctc ctc cca ggg tcc agc gcc tgt gct gga aac agc agc cag 3206

Glu Glu Leu Leu Pro Gly Ser Ser Ala Cys Ala Gly Asn Ser Ser Gln

990 995 1000

agc cgc ccc tgc ccc tac agc gag att ccc gtc atc ctg cca gcc 3251

Ser Arg Pro Cys Pro Tyr Ser Glu Ile Pro Val Ile Leu Pro Ala

1005 1010 1015

tcc agc atg gag gag gcc acc gac tgt gca ggt aaa aga aac cgg 3296

Ser Ser Met Glu Glu Ala Thr Asp Cys Ala Gly Lys Arg Asn Arg

1020 1025 1030

acc tac ctc atg ctg cgg tcc tcc cag ccc tcc agc acc cca ctc 3341

Thr Tyr Leu Met Leu Arg Ser Ser Gln Pro Ser Ser Thr Pro Leu

1035 1040 1045

caa agt ctg gac tct ttc cac atc ctg ctc cag aca gcc aag ctt 3386

Gln Ser Leu Asp Ser Phe His Ile Leu Leu Gln Thr Ala Lys Leu

1050 1055 1060

tgt tgg ggt ccc cac tgc ttt gag atg ggt tca atc tca tcc act 3431

Cys Trp Gly Pro His Cys Phe Glu Met Gly Ser Ile Ser Ser Thr

1065 1070 1075

tgg tgg cca cgg gca tct cct gct tct tgg gct ctg ggc tcc tga 3476

Trp Trp Pro Arg Ala Ser Pro Ala Ser Trp Ala Leu Gly Ser

1080 1085 1090

ccctagcagt gtacctgtct tgccagcact gccagcgtca gtcccaggag tccacactgg 3536

tccatcctgc cacccccaac catttgcact acaagggcgg aggcaccccg aagaatgaaa 3596

agtacacacc catggaattc aagaccctga acaagaataa cttgatccct gatgacagag 3656

ccaacttcta cccattgcag cagaccaatg tgtacacgac tacttactac ccaagccccc 3716

tgaacaaaca cagcttccgg cccgaggcct cacctggaca acggtgcttc cccaacagct 3776

gataccgccg tcctggggac ttgggcttct tgccttcata aggcacagag cagatggaga 3836

tgggacagtg gagccagttt ggttttctcc ctctgcacta ggccaagaac ttgctgcctt 3896

gcctgtgggg ggtcccatcc ggcttcagag agctctggct ggcattgacc atgggggaaa 3956

gggctggttt caggctgaca tatggccgca ggtccagttc agcccaggtc tctcatggtt 4016

atcttccaac ccactgtcac gctgacacta tgctgccatg cctgggctgt ggacctactg 4076

ggcatttgag gaattggaga atggagatgg caagagggca ggcttttaag tttgggttgg 4136

agacaacttc ctgtggcccc cacaagctga gtctggcctt ctccagctgg ccccaaaaaa 4196

ggcctttgct acatcctgat tatctctgaa agtaatcaat caagtggctc cagtagctct 4256

ggattttctg ccagggctgg gccattgtgg tgctgcccca gtatgacatg ggaccaaggc 4316

cagcgcaggt tatccacctc tgcctggaag tctatactct acccagggca tccctctggt 4376

cagaggcagt gagtactggg aactggaggc tgacctgtgc ttagaagtcc tttaatctgg 4436

gctggtacag gcctcagcct tgccctcaat gcacgaaagg tggcccagga gagaggatca 4496

atgccatagg aggcagaagt ctggcctctg tgcctctatg gagactatct tccagttgct 4556

gctcaacaga gttgttggct gagacctgct tgggagtctc tgctggccct tcatctgttc 4616

aggaacacac acacacacac actcacacac gcacacacaa tcacaatttg ctacagcaac 4676

aaaaaagaca ttgggctgtg gcattattaa ttaaagatga tatccagtc 4725

<210>64

<211>1092

<212>PRT

＜213〉people (Homo sapiens)

<400>64

Met Pro Cys Gly Phe Ser Pro Ser Pro Val Ala His His Leu Val Pro

1 5 10 15

Gly Pro Pro Asp Thr Pro Ala Gln Gln Leu Arg Cys Gly Trp Thr Val

20 25 30

Gly Gly Trp Leu Leu Ser Leu Val Arg Gly Leu Leu Pro Cys Leu Pro

35 40 45

Pro Gly Ala Arg Thr Ala Glu Gly Pro Ile Met Val Leu Ala Gly Pro

50 55 60

Leu Ala Val Ser Leu Leu Leu Pro Ser Leu Thr Leu Leu Val Ser His

65 70 75 80

Leu Ser Ser Ser Gln Asp Val Ser Ser Glu Pro Ser Ser Glu Gln Gln

85 90 95

Leu Cys Ala Leu Ser Lys His Pro Thr Val Ala Phe Glu Asp Leu Gln

100 105 110

Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly Ala Arg Asp Phe Ser Gln

115 120 125

Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu Ile Val Gly Ala Arg Asn

130 135 140

Tyr Leu Phe Arg Leu Ser Leu Ala Asn Val Ser Leu Leu Gln Ala Thr

145 150 155 160

Glu Trp Ala Ser Ser Glu Asp Thr Arg Arg Ser Cys Gln Ser Lys Gly

165 170 175

Lys Thr Glu Glu Glu Cys Gln Asn Tyr Val Arg Val Leu Ile Val Ala

180 185 190

Gly Arg Lys Val Phe Met Cys Gly Thr Asn Ala Phe Ser Pro Met Cys

195 200 205

Thr Ser Arg Gln Val Gly Asn Leu Ser Arg Thr Ile Glu Lys Ile Asn

210 215 220

Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg His Asn Ser Thr Ala Val

225 230 235 240

Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala Thr Val Ile Asp Phe Ser

245 250 255

Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu Gly Ser Gly Pro Pro Leu

260 265 270

Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu Asn Glu Pro Asn Phe Val

275 280 285

Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe Phe Leu Arg Glu Asn

290 295 300

Ala Val Glu His Asp Cys Gly Arg Thr Val Tyr Ser Arg Val Ala Arg

305 310 315 320

Val Cys Lys Asn Asp Val Gly Gly Arg Phe Leu Leu Glu Asp Thr Trp

325 330 335

Thr Thr Phe Met Lys Ala Arg Leu Asn Cys Ser Arg Pro Gly Glu Val

340 345 350

Pro Phe Tyr Tyr Asn Glu Leu Gln Ser Ala Phe His Leu Pro Glu Gln

355 360 365

Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn Val Asn Ser Ile Ala Ala

370 375 380

Ser Ala Val Cys Ala Phe Asn Leu Ser Ala Ile Ser Gln Ala Phe Asn

385 390 395 400

Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg Ala Ala Trp Leu Pro Ile

405 410 415

Ala Asn Pro Ile Pro Asn Phe Gln Cys Gly Thr Leu Pro Glu Thr Gly

420 425 430

Pro Asn Glu Asn Leu Thr Glu Arg Ser Leu Gln Asp Ala Gln Arg Leu

435 440 445

Phe Leu Met Ser Glu Ala Val Gln Pro Val Thr Pro Glu Pro Cys Val

450 455 460

Thr Gln Asp Ser Val Arg Phe Ser His Leu Val Val Asp Leu Val Gln

465 470 475 480

Ala Lys Asp Thr Leu Tyr His Val Leu Tyr Ile Gly Thr Glu Ser Gly

485 490 495

Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser Arg Ser Leu His Gly Cys

500 505 510

Tyr Leu Glu Glu Leu His Val Leu Pro Pro Gly Arg Arg Glu Pro Leu

515 520 525

Arg Ser Leu Arg Ile Leu His Ser Ala Arg Ala Leu Phe Val Gly Leu

530 535 540

Arg Asp Gly Val Leu Arg Val Pro Leu Glu Arg Cys Ala Ala Tyr Arg

545 550 555 560

Ser Gln Gly Ala Cys Leu Gly Ala Arg Asp Pro Tyr Cys Gly Trp Asp

565 570 575

Gly Lys Gln Gln Arg Cys Ser Thr Leu Glu Asp Ser Ser Asn Met Ser

580 585 590

Leu Trp Thr Gln Asn Ile Thr Ala Cys Pro Val Arg Asn Val Thr Arg

595 600 605

Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp Gln Pro Cys Glu His Leu

610 615 620

Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys Arg Ala Arg Ser Cys Asp

625 630 635 640

Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp Cys Leu Gly Pro Ala Ile

645 650 655

His Ile Ala Asn Cys Ser Arg Asn Gly Ala Trp Thr Pro Trp Ser Ser

660 665 670

Trp Ala Leu Cys Ser Thr Ser Cys Gly Ile Gly Phe Gln Val Arg Gln

675 680 685

Arg Ser Cys Ser Asn Pro Ala Pro Arg His Gly Gly Arg Ile Phe Val

690 695 700

Gly Lys Ser Arg Glu Glu Arg Phe Cys Asn Glu Asn Thr Pro Cys Pro

705 710 715 720

Val Pro Ile Phe Trp Ala Ser Trp Gly Ser Trp Ser Lys Cys Ser Ser

725 730 735

Asn Cys Gly Gly Gly Met Gln Ser Arg Arg Arg Ala Cys Glu Asn Gly

740 745 750

Asn Ser Cys Leu Gly Cys Gly Glu Phe Lys Thr Cys Asn Pro Glu Gly

755 760 765

Cys Pro Glu Val Arg Arg Asn Thr Pro Trp Thr Pro Trp Leu Pro Val

770 775 780

Asn Val Thr Gln Gly Gly Ala Arg Gln Glu Gln Arg Phe Arg Phe Thr

785 790 795 800

Cys Arg Ala Pro Leu Ala Asp Pro His Gly Leu Gln Phe Gly Arg Arg

805 810 815

Arg Thr Glu Thr Arg Thr Cys Pro Ala Asp Gly Ser Gly Ser Cys Asp

820 825 830

Thr Asp Ala Leu Val Glu Val Leu Leu Arg Ser Gly Ser Thr Ser Pro

835 840 845

His Thr Val Ser Gly Gly Trp Ala Ala Trp Gly Pro Trp Ser Ser Cys

850 855 860

Ser Arg Asp Cys Glu Leu Gly Phe Arg Val Arg Lys Arg Thr Cys Thr

865 870 875 880

Asn Pro Glu Pro Arg Asn Gly Gly Leu Pro Cys Val Gly Asp Ala Ala

885 890 895

Glu Tyr Gln Asp Cys Asn Pro Gln Ala Cys Pro Val Arg Gly Ala Trp

900 905 910

Ser Cys Trp Thr Ser Trp Ser Pro Cys Ser Ala Ser Cys Gly Gly Gly

915 920 925

His Tyr Gln Arg Thr Arg Ser Cys Thr Ser Pro Ala Pro Ser Pro Gly

930 935 940

Glu Asp Ile Cys Leu Gly Leu His Thr Glu Glu Ala Leu Cys Ala Thr

945 950 955 960

Gln Ala Cys Pro Gly Trp Ser Pro Trp Ser Glu Trp Ser Lys Cys Thr

965 970 975

Asp Asp Gly Ala Gln Ser Arg Ser Arg His Cys Glu Glu Leu Leu Pro

980 985 990

Gly Ser Ser Ala Cys Ala Gly Asn Ser Ser Gln Ser Arg Pro Cys Pro

995 1000 1005

Tyr Ser Glu Ile Pro Val Ile Leu Pro Ala Ser Ser Met Glu Glu

1010 1015 1020

Ala Thr Asp Cys Ala Gly Lys Arg Asn Arg Thr Tyr Leu Met Leu

1025 1030 1035

Arg Ser Ser Gln Pro Ser Ser Thr Pro Leu Gln Ser Leu Asp Ser

1040 1045 1050

Phe His Ile Leu Leu Gln Thr Ala Lys Leu Cys Trp Gly Pro His

1055 1060 1065

Cys Phe Glu Met Gly Ser Ile Ser Ser Thr Trp Trp Pro Arg Ala

1070 1075 1080

Ser Pro Ala Ser Trp Ala Leu Gly Ser

1085 1090

<210>65

<211>4494

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(71)..(3526)

<400>65

gggaaccggg cacctgcacc cgcctctggg agtgagtggc tccagctggt gcctggcctg 60

tgtctcttgg atg ccc tgt ggc ttc agt ccg tct cct gtt gcc cac cac 109

Met Pro Cys Gly Phe Ser Pro Ser Pro Val Ala His His

1 5 10

ctc gtc cct ggg ccg cct gat acc cca gcc caa cag cta agg tgt gga 157

Leu Val Pro Gly Pro Pro Asp Thr Pro Ala Gln Gln Leu Arg Cys Gly

15 20 25

tgg aca gta ggg ggc tgg ctt ctc tca ctg gtc agg ggt ctt ctc ccc 205

Trp Thr Val Gly Gly Trp Leu Leu Ser Leu Val Arg Gly Leu Leu Pro

30 35 40 45

tgt ctg cct ccc gga gct agg act gca gag ggg cct atc atg gtg ctt 253

Cys Leu Pro Pro Gly Ala Arg Thr Ala Glu Gly Pro Ile Met Val Leu

50 55 60

gca ggc ccc ctg gct gtc tcg ctg ttg ctg ccc agc ctc aca ctg ctg 301

Ala Gly Pro Leu Ala Val Ser Leu Leu Leu Pro Ser Leu Thr Leu Leu

65 70 75

gtg tcc cac ctc tcc agc tcc cag gat gtc tcc agt gag ccc agc agt 349

Val Ser His Leu Ser Ser Ser Gln Asp Val Ser Ser Glu Pro Ser Ser

80 85 90

gag cag cag ctg tgc gcc ctt agc aag cac ccc acc gtg gcc ttt gaa 397

Glu Gln Gln Leu Cys Ala Leu Ser Lys His Pro Thr Val Ala Phe Glu

95 100 105

gac ctg cag ccg tgg gtc tct aac ttc acc tac cct gga gcc cgg gat 445

Asp Leu Gln Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly Ala Arg Asp

110 115 120 125

ttc tcc cag ctg gct ttg gac ccc tcc ggg aac cag ctc atc gtg gga 493

Phe Ser Gln Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu Ile Val Gly

130 135 140

gcc agg aac tac ctc ttc aga ctc agc ctt gcc aat gtc tct ctt ctt 541

Ala Arg Asn Tyr Leu Phe Arg Leu Ser Leu Ala Asn Val Ser Leu Leu

145 150 155

cag gcc aca gag tgg gcc tcc agt gag gac acg cgc cgc tcc tgc caa 589

Gln Ala Thr Glu Trp Ala Ser Ser Glu Asp Thr Arg Arg Ser Cys Gln

160 165 170

agc aaa ggg aag act gag gag gag tgt cag aac tac gtg cga gtc ctg 637

Ser Lys Gly Lys Thr Glu Glu Glu Cys Gln Asn Tyr Val Arg Val Leu

175 180 185

atc gtc gcc ggc cgg aag gtg ttc atg tgt gga acc aat gcc ttt tcc 685

Ile Val Ala Gly Arg Lys Val Phe Met Cys Gly Thr Asn Ala Phe Ser

190 195 200 205

ccc atg tgc acc agc aga cag gtg ggg aac ctc agc cgg act att gag 733

Pro Met Cys Thr Ser Arg Gln Val Gly Asn Leu Ser Arg Thr Ile Glu

210 215 220

aag atc aat ggt gtg gcc cgc tgc ccc tat gac cca cgc cac aac tcc 781

Lys Ile Asn Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg His Asn Ser

225 230 235

aca gct gtc atc tcc tcc cag ggg gag ctc tat gca gcc acg gtc atc 829

Thr Ala Val Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala Thr Val Ile

240 245 250

gac ttc tca ggt cgg gac cct gcc atc tac cgc agc ctg ggc agt ggg 877

Asp Phe Ser Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu Gly Ser Gly

255 260 265

cca ccg ctt cgc act gcc caa tat aac tcc aag tgg ctt aat gag cca 925

Pro Pro Leu Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu Asn Glu Pro

270 275 280 285

aac ttc gtg gca gcc tat gat att ggg ctg ttt gca tac ttc ttc ctg 973

Asn Phe Val Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe Phe Leu

290 295 300

cgg gag aac gca gtg gag cac gac tgt gga cgc acc gtg tac tct cgc 1021

Arg Glu Asn Ala Val Glu His Asp Cys Gly Arg Thr Val Tyr Ser Arg

305 310 315

gtg gcc cgc gtg tgc aag aat gac gtg ggg ggc cga ttc ctg ctg gag 1069

Val Ala Arg Val Cys Lys Asn Asp Val Gly Gly Arg Phe Leu Leu Glu

320 325 330

gac aca tgg acc aca ttc atg aag gcc cgg ctc aac tgc tcc cgc ccg 1117

Asp Thr Trp Thr Thr Phe Met Lys Ala Arg Leu Asn Cys Ser Arg Pro

335 340 345

ggc gag gtc ccc ttc tac tat aac gag ctg cag agt gcc ttc cac ttg 1165

Gly Glu Val Pro Phe Tyr Tyr Asn Glu Leu Gln Ser Ala Phe His Leu

350 355 360 365

ccg gag cag gac ctc atc tat gga gtt ttc aca acc aac gta aac agc 1213

Pro Glu Gln Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn Val Asn Ser

370 375 380

att gcg gct tct gct gtc tgc gcc ttc aac ctc agt gct atc tcc cag 1261

Ile Ala Ala Ser Ala Val Cys Ala Phe Asn Leu Ser Ala Ile Ser Gln

385 390 395

gct ttc aat ggc cca ttt cgc tac cag gag aac ccc agg gct gcc tgg 1309

Ala Phe Asn Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg Ala Ala Trp

400 405 410

ctc ccc ata gcc aac ccc atc ccc aat ttc cag tgt ggc acc ctg cct 1357

Leu Pro Ile Ala Asn Pro Ile Pro Asn Phe Gln Cys Gly Thr Leu Pro

415 420 425

gag acc ggt ccc aac gag aac ctg acg gag cgc agc ctg cag gac gcg 1405

Glu Thr Gly Pro Asn Glu Asn Leu Thr Glu Arg Ser Leu Gln Asp Ala

430 435 440 445

cag cgc ctc ttc ctg atg agc gag gcc gtg cag ccg gtg aca ccc gag 1453

Gln Arg Leu Phe Leu Met Ser Glu Ala Val Gln Pro Val Thr Pro Glu

450 455 460

ccc tgt gtc acc cag gac agc gtg cgc ttc tca cac ctc gtg gtg gac 1501

Pro Cys Val Thr Gln Asp Ser Val Arg Phe Ser His Leu Val Val Asp

465 470 475

ctg gtg cag gct aaa gac acg ctc tac cat gta ctc tac att ggc acc 1549

Leu Val Gln Ala Lys Asp Thr Leu Tyr His Val Leu Tyr Ile Gly Thr

480 485 490

gag tcg ggc acc atc ctg aag gcg ctg tcc acg gcg agc cgc agc ctc 1597

Glu Ser Gly Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser Arg Ser Leu

495 500 505

cac ggc tgc tac ctg gag gag ctg cac gtg ctg ccc ccc ggg cgc cgc 1645

His Gly Cys Tyr Leu Glu Glu Leu His Val Leu Pro Pro Gly Arg Arg

510 515 520 525

gag ccc ctg cgc agc ctg cgc atc ctg cac agc gcc cgc gcg ctc ttc 1693

Glu Pro Leu Arg Ser Leu Arg Ile Leu His Ser Ala Arg Ala Leu Phe

530 535 540

gtg ggg ctg aga gac ggc gtc ctg cgg gtc cca ctg gag agg tgc gcc 1741

Val Gly Leu Arg Asp Gly Val Leu Arg Val Pro Leu Glu Arg Cys Ala

545 550 555

gcc tac cgc agc cag ggg gca tgc ctg ggg gcc cgg gac ccg tac tgt 1789

Ala Tyr Arg Ser Gln Gly Ala Cys Leu Gly Ala Arg Asp Pro Tyr Cys

560 565 570

ggc tgg gac ggg aag cag caa cgt tgc agc aca ctc gag gac agc tcc 1837

Gly Trp Asp Gly Lys Gln Gln Arg Cys Ser Thr Leu Glu Asp Ser Ser

575 580 585

aac atg agc ctc tgg acc cag aac atc acc gcc tgt cct gtg cgg aat 1885

Asn Met Ser Leu Trp Thr Gln Asn Ile Thr Ala Cys Pro Val Arg Asn

590 595 600 605

gtg aca cgg gat ggg ggc ttc ggc cca tgg tca cca tgg caa cca tgt 1933

Val Thr Arg Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp Gln Pro Cys

610 615 620

gag cac ttg gat ggg gac aac tca ggc tct tgc ctg tgt cga gct cga 1981

Glu His Leu Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys Arg Ala Arg

625 630 635

tcc tgt gat tcc cct cga ccc cgc tgt ggg ggc ctt gac tgc ctg ggg 2029

Ser Cys Asp Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp Cys Leu Gly

640 645 650

cca gcc atc cac atc gcc aac tgc tcc agg aat ggg gcg tgg acc ccg 2077

Pro Ala Ile His Ile Ala Asn Cys Ser Arg Asn Gly Ala Trp Thr Pro

655 660 665

tgg tca tcg tgg gcg ctg tgc agc acg tcc tgt ggc atc ggc ttc cag 2125

Trp Ser Ser Trp Ala Leu Cys Ser Thr Ser Cys Gly Ile Gly Phe Gln

670 675 680 685

gtc cgc cag cga agt tgc agc aac cct gct ccc cgc cac ggg ggc cgc 2173

Val Arg Gln Arg Ser Cys Ser Asn Pro Ala Pro Arg His Gly Gly Arg

690 695 700

atc tgc gtg ggc aag agc cgg gag gaa cgg ttc tgt aat gag aac acg 2221

Ile Cys Val Gly Lys Ser Arg Glu Glu Arg Phe Cys Asn Glu Asn Thr

705 710 715

cct tgc ccg gtg ccc atc ttc tgg gct tcc tgg ggc tcc tgg agc aag 2269

Pro Cys Pro Val Pro Ile Phe Trp Ala Ser Trp Gly Ser Trp Ser Lys

720 725 730

tgc agc agc aac tgt gga ggg ggc atg cag tcg cgg cgt cgg gcc tgc 2317

Cys Ser Ser Asn Cys Gly Gly Gly Met Gln Ser Arg Arg Arg Ala Cys

735 740 745

gag aac ggc aac tcc tgc ctg ggc tgc ggc gtg gag ttc aag acg tgc 2365

Glu Asn Gly Asn Ser Cys Leu Gly Cys Gly Val Glu Phe Lys Thr Cys

750 755 760 765

aac ccc gag ggc tgc ccc gaa gtg cgg cgc aac acc ccc tgg acg ccg 2413

Asn Pro Glu Gly Cys Pro Glu Val Arg Arg Asn Thr Pro Trp Thr Pro

770 775 780

tgg ctg ccc gtg aac gtg acg cag ggc ggg gca cgg cag gag cag cgg 2461

Trp Leu Pro Val Asn Val Thr Gln Gly Gly Ala Arg Gln Glu Gln Arg

785 790 795

ttc cgc ttc acc tgc cgc gcg ccc ctt gca gac ccg cac ggc ctg cag 2509

Phe Arg Phe Thr Cys Arg Ala Pro Leu Ala Asp Pro His Gly Leu Gln

800 805 810

ttc ggc agg aga agg acc gag acg agg acc tgt ccc gcg gac ggc tcc 2557

Phe Gly Arg Arg Arg Thr Glu Thr Arg Thr Cys Pro Ala Asp Gly Ser

815 820 825

ggc tcc tgc gac acc gac gcc ctg gtg gag gac ctc ctg cgc agc ggg 2605

Gly Ser Cys Asp Thr Asp Ala Leu Val Glu Asp Leu Leu Arg Ser Gly

830 835 840 845

agc acc tcc ccg cac acg gtg agc ggg ggc tgg gcc gcc tgg ggc ccg 2653

Ser Thr Ser Pro His Thr Val Ser Gly Gly Trp Ala Ala Trp Gly Pro

850 855 860

tgg tcg tcc tgc tcc cgg gac tgc gag ctg ggc ttc cgc gtc cgc aag 2701

Trp Ser Ser Cys Ser Arg Asp Cys Glu Leu Gly Phe Arg Val Arg Lys

865 870 875

aga acg tgc act aac ccg gag ccc cgc aac ggg ggc ctg ccc tgc gtg 2749

Arg Thr Cys Thr Asn Pro Glu Pro Arg Asn Gly Gly Leu Pro Cys Val

880 885 890

ggc gat gct gcc gag tac cag gac tgc aac ccc cag gct tgc cca gtt 2797

Gly Asp Ala Ala Glu Tyr Gln Asp Cys Asn Pro Gln Ala Cys Pro Val

895 900 905

cgg ggt gct tgg tcc tgc tgg acc tca tgg tct cca tgc tca gct tcc 2845

Arg Gly Ala Trp Ser Cys Trp Thr Ser Trp Ser Pro Cys Ser Ala Ser

910 915 920 925

tgt ggt ggg ggt cac tat caa cgc acc cgt tcc tgc acc agc ccc gca 2893

Cys Gly Gly Gly His Tyr Gln Arg Thr Arg Ser Cys Thr Ser Pro Ala

930 935 940

ccc tcc cca ggt gag gac atc tgt ctc ggg ctg cac acg gag gag gca 2941

Pro Ser Pro Gly Glu Asp Ile Cys Leu Gly Leu His Thr Glu Glu Ala

945 950 955

cta tgt gcc aca cag gcc tgc cca gaa ggc tgg tcg ccc tgg tct gag 2989

Leu Cys Ala Thr Gln Ala Cys Pro Glu Gly Trp Ser Pro Trp Ser Glu

960 965 970

tgg agt aag tgc act gac gac gga gcc cag agc cga agc cgg cac tgt 3037

Trp Ser Lys Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser Arg His Cys

975 980 985

gag gag ctc ctc cca ggg tcc agc gcc tgt gct gga aac agc agc cag 3085

Glu Glu Leu Leu Pro Gly Ser Ser Ala Cys Ala Gly Asn Ser Ser Gln

990 995 1000 1005

agc cgc ccc tgc ccc tac agc gag att ccc gtc atc ctg cca gcc 3130

Ser Arg Pro Cys Pro Tyr Ser Glu Ile Pro Val Ile Leu Pro Ala

1010 1015 1020

tcc agc atg gag gag gcc acc ggc tgt gca ggg ttc aat ctc atc 3175

Ser Ser Met Glu Glu Ala Thr Gly Cys Ala Gly Phe Asn Leu Ile

1025 1030 1035

cac ttg gtg gcc acg ggc atc tcc tgc ttc ttg ggc tct ggg ctc 3220

His Leu Val Ala Thr Gly Ile Ser Cys Phe Leu Gly Ser Gly Leu

1040 1045 1050

ctg acc cta gca gtg tac ctg tct tgc cag cac tgc cag cgt cag 3265

Leu Thr Leu Ala Val Tyr Leu Ser Cys Gln His Cys Gln Arg Gln

1055 1060 1065

tcc cag gag tcc aca ctg gtc cat cct gcc acc ccc aac cat ttg 3310

Ser Gln Glu Ser Thr Leu Val His Pro Ala Thr Pro Asn His Leu

1070 1075 1080

cac tac aag ggc gga ggc acc ccg aag aat gaa aag tac aca ccc 3355

His Tyr Lys Gly Gly Gly Thr Pro Lys Asn Glu Lys Tyr Thr Pro

1085 1090 1095

atg gaa ttc aag acc ctg aac aag aat aac ttg atc cct gat gac 3400

Met Glu Phe Lys Thr Leu Asn Lys Asn Asn Leu Ile Pro Asp Asp

1100 1105 1110

aga gcc aac ttc tac cca ttg cag cag acc aat gtg tac acg act 3445

Arg Ala Asn Phe Tyr Pro Leu Gln Gln Thr Asn Val Tyr Thr Thr

1115 1120 1125

act tac tac cca agc ccc ctg aac aaa cac agc ttc cgg ccc gag 3490

Thr Tyr Tyr Pro Ser Pro Leu Asn Lys His Ser Phe Arg Pro Glu

1130 1135 1140

gcc tca cct gga caa cgg tgc ttc ccc aac agc tga taccgccgtc 3536

Ala Ser Pro Gly Gln Arg Cys Phe Pro Asn Ser

1145 1150

ctggggactt gggettcttg ccttcataag gcacagagca gatggagatg ggacagtgga 3596

gccagtttgg ttttctccct ctgcactagg ccaagaactt gctgccttgc ctgtgggggg 3656

tcccatccgg cttcagagag ctctggctgg cattgaccat gggggaaagg gctggtttca 3716

ggctgacata tggccgcagg tccagttcag cccaggtctc tcatggttat cttccaaccc 3776

actgtcacgc tgacactatg ctgccatgcc tgggctgtgg acctactggg catttgagga 3836

attggagaat ggagatggca agagggcagg cttttaagtt tgggttggag acaacttcct 3896

gtggccccca caagctgagt ctggccttct ccagctggcc ccaaaaaagg cctttgctac 3956

atcctgatta tctctgaaag taatcaatca agtggctcca gtagctctgg attttctgcc 4016

agggctgggc cattgtggtg ctgccccagt atgacatggg accaaggcca gcgcaggtta 4076

ttcacctctg cctggaagtc tatactctac ccagggcatc cctctggtca gaggcagtga 4136

gtactgggaa ctggaggctg acctgtgctt agaagtcctt taatctgggc tggtacaggc 4196

ctcagccttg ccctcaatgc acgaaaggtg gcccaggaga gaggatcaat gccataggag 4256

gcagaagtct ggcctctgtg cctctatgga gactatcttc cagttgctgc tcaacagagt 4316

tgttggctga gacctgcttg ggagtctctg ctggcccttc atctgttcag gaacacacac 4376

acacacacac tcacacacgc acacacaatc acaatttgct acagcaacaa aaaagacatt 4436

gggctgtggc attattaatt aaagatgata tccagtctca aaaaaaaaaa aaaaaaaa 4494

<210>66

<211>1151

<212>PRT

＜213〉people (Homo sapiens)

<400>66

Met Pro Cys Gly Phe Ser Pro Ser Pro Val Ala His His Leu Val Pro

1 5 10 15

Gly Pro Pro Asp Thr Pro Ala Gln Gln Leu Arg Cys Gly Trp Thr Val

20 25 30

Gly Gly Trp Leu Leu Ser Leu Val Arg Gly Leu Leu Pro Cys Leu Pro

35 40 45

Pro Gly Ala Arg Thr Ala Glu Gly Pro Ile Met Val Leu Ala Gly Pro

50 55 60

Leu Ala Val Ser Leu Leu Leu Pro Ser Leu Thr Leu Leu Val Ser His

65 70 75 80

Leu Ser Ser Ser Gln Asp Val Ser Ser Glu Pro Ser Ser Glu Gln Gln

85 90 95

Leu Cys Ala Leu Ser Lys His Pro Thr Val Ala Phe Glu Asp Leu Gln

100 105 110

Pro Trp Val Ser Asn Phe Thr Tyr Pro Gly Ala Arg Asp Phe Ser Gln

115 120 125

Leu Ala Leu Asp Pro Ser Gly Asn Gln Leu Ile Val Gly Ala Arg Asn

130 135 140

Tyr Leu Phe Arg Leu Ser Leu Ala Asn Val Ser Leu Leu Gln Ala Thr

145 150 155 160

Glu Trp Ala Ser Ser Glu Asp Thr Arg Arg Ser Cys Gln Ser Lys Gly

165 170 175

Lys Thr Glu Glu Glu Cys Gln Asn Tyr Val Arg Val Leu Ile Val Ala

180 185 190

Gly Arg Lys Val Phe Met Cys Gly Thr Asn Ala Phe Ser Pro Met Cys

195 200 205

Thr Ser Arg Gln Val Gly Asn Leu Ser Arg Thr Ile Glu Lys Ile Asn

210 215 220

Gly Val Ala Arg Cys Pro Tyr Asp Pro Arg His Asn Ser Thr Ala Val

225 230 235 240

Ile Ser Ser Gln Gly Glu Leu Tyr Ala Ala Thr Val Ile Asp Phe Ser

245 250 255

Gly Arg Asp Pro Ala Ile Tyr Arg Ser Leu Gly Ser Gly Pro Pro Leu

260 265 270

Arg Thr Ala Gln Tyr Asn Ser Lys Trp Leu Asn Glu Pro Asn Phe Val

275 280 285

Ala Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe Phe Leu Arg Glu Asn

290 295 300

Ala Val Glu His Asp Cys Gly Arg Thr Val Tyr Ser Arg Val Ala Arg

305 310 315 320

Val Cys Lys Asn Asp Val Gly Gly Arg Phe Leu Leu Glu Asp Thr Trp

325 330 335

Thr Thr Phe Met Lys Ala Arg Leu Asn Cys Ser Arg Pro Gly Glu Val

340 345 350

Pro Phe Tyr Tyr Asn Glu Leu Gln Ser Ala Phe His Leu Pro Glu Gln

355 360 365

Asp Leu Ile Tyr Gly Val Phe Thr Thr Asn Val Asn Ser Ile Ala Ala

370 375 380

Ser Ala Val Cys Ala Phe Asn Leu Ser Ala Ile Ser Gln Ala Phe Asn

385 390 395 400

Gly Pro Phe Arg Tyr Gln Glu Asn Pro Arg Ala Ala Trp Leu Pro Ile

405 410 415

Ala Asn Pro Ile Pro Asn Phe Gln Cys Gly Thr Leu Pro Glu Thr Gly

420 425 430

Pro Asn Glu Asn Leu Thr Glu Arg Ser Leu Gln Asp Ala Gln Arg Leu

435 440 445

Phe Leu Met Ser Glu Ala Val Gln Pro Val Thr Pro Glu Pro Cys Val

450 455 460

Thr Gln Asp Ser Val Arg Phe Ser His Leu Val Val Asp Leu Val Gln

465 470 475 480

Ala Lys Asp Thr Leu Tyr His Val Leu Tyr Ile Gly Thr Glu Ser Gly

485 490 495

Thr Ile Leu Lys Ala Leu Ser Thr Ala Ser Arg Ser Leu His Gly Cys

500 505 510

Tyr Leu Glu Glu Leu His Val Leu Pro Pro Gly Arg Arg Glu Pro Leu

515 520 525

Arg Ser Leu Arg Ile Leu His Ser Ala Arg Ala Leu Phe Val Gly Leu

530 535 540

Arg Asp Gly Val Leu Arg Val Pro Leu Glu Arg Cys Ala Ala Tyr Arg

545 550 555 560

Ser Gln Gly Ala Cys Leu Gly Ala Arg Asp Pro Tyr Cys Gly Trp Asp

565 570 575

Gly Lys Gln Gln Arg Cys Ser Thr Leu Glu Asp Ser Ser Asn Met Ser

580 585 590

Leu Trp Thr Gln Asn Ile Thr Ala Cys Pro Val Arg Asn Val Thr Arg

595 600 605

Asp Gly Gly Phe Gly Pro Trp Ser Pro Trp Gln Pro Cys Glu His Leu

610 615 620

Asp Gly Asp Asn Ser Gly Ser Cys Leu Cys Arg Ala Arg Ser Cys Asp

625 630 635 640

Ser Pro Arg Pro Arg Cys Gly Gly Leu Asp Cys Leu Gly Pro Ala Ile

645 650 655

His Ile Ala Asn Cys Ser Arg Asn Gly Ala Trp Thr Pro Trp Ser Ser

660 665 670

Trp Ala Leu Cys Ser Thr Ser Cys Gly Ile Gly Phe Gln Va1 Arg Gln

675 680 685

Arg Ser Cys Ser Asn Pro Ala Pro Arg His Gly Gly Arg Ile Cys Val

690 695 700

Gly Lys Ser Arg Glu Glu Arg Phe Cys Asn Glu Asn Thr Pro Cys Pro

705 710 715 720

Val Pro Ile Phe Trp Ala Ser Trp Gly Ser Trp Ser Lys Cys Ser Ser

725 730 735

Asn Cys Gly Gly Gly Met Gln Ser Arg Arg Arg Ala Cys Glu Asn Gly

740 745 750

Asn Ser Cys Leu Gly Cys Gly Val Glu Phe Lys Thr Cys Asn Pro Glu

755 760 765

Gly Cys Pro Glu Val Arg Arg Asn Thr Pro Trp Thr Pro Trp Leu Pro

770 775 780

Val Asn Val Thr Gln Gly Gly Ala Arg Gln Glu Gln Arg Phe Arg Phe

785 790 795 800

Thr Cys Arg Ala Pro Leu Ala Asp Pro His Gly Leu Gln Phe Gly Arg

805 810 815

Arg Arg Thr Glu Thr Arg Thr Cys Pro Ala Asp Gly Ser Gly Ser Cys

820 825 830

Asp Thr Asp Ala Leu Val Glu Asp Leu Leu Arg Ser Gly Ser Thr Ser

835 840 845

Pro His Thr Val Ser Gly Gly Trp Ala Ala Trp Gly Pro Trp Ser Ser

850 855 860

Cys Ser Arg Asp Cys Glu Leu Gly Phe Arg Val Arg Lys Arg Thr Cys

865 870 875 880

Thr Asn Pro Glu Pro Arg Asn Gly Gly Leu Pro Cys Val Gly Asp Ala

885 890 895

Ala Glu Tyr Gln Asp Cys Asn Pro Gln Ala Cys Pro Val Arg Gly Ala

900 905 910

Trp Ser Cys Trp Thr Ser Trp Ser Pro Cys Ser Ala Ser Cys Gly Gly

915 920 925

Gly His Tyr Gln Arg Thr Arg Ser Cys Thr Ser Pro Ala Pro Ser Pro

930 935 940

Gly Glu Asp Ile Cys Leu Gly Leu His Thr Glu Glu Ala Leu Cys Ala

945 950 955 960

Thr Gln Ala Cys Pro Glu Gly Trp Ser Pro Trp Ser Glu Trp Ser Lys

965 970 975

Cys Thr Asp Asp Gly Ala Gln Ser Arg Ser Arg His Cys Glu Glu Leu

980 985 990

Leu Pro Gly Ser Ser Ala Cys Ala Gly Asn Ser Ser Gln Ser Arg Pro

995 1000 1005

Cys Pro Tyr Ser Glu Ile Pro Val Ile Leu Pro Ala Ser Ser Met

1010 1015 1020

Glu Glu Ala Thr Gly Cys Ala Gly Phe Asn Leu Ile His Leu Val

1025 1030 1035

Ala Thr Gly Ile Ser Cys Phe Leu Gly Ser Gly Leu Leu Thr Leu

1040 1045 1050

Ala Val Tyr Leu Ser Cys Gln His Cys Gln Arg Gln Ser Gln Glu

1055 1060 1065

Ser Thr Leu Val His Pro Ala Thr Pro Asn His Leu His Tyr Lys

1070 1075 1080

Gly Gly Gly Thr Pro Lys Asn Glu Lys Tyr Thr Pro Met Glu Phe

1085 1090 1095

Lys Thr Leu Asn Lys Asn Asn Leu Ile Pro Asp Asp Arg Ala Asn

1100 1105 1110

Phe Tyr Pro Leu Gln Gln Thr Asn Val Tyr Thr Thr Thr Tyr Tyr

1115 1120 1125

Pro Ser Pro Leu Asn Lys His Ser Phe Arg Pro Glu Ala Ser Pro

1130 1135 1140

Gly Gln Arg Cys Phe Pro Asn Ser

1145 1150

<210>67

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>67

cctgaagggt gcctagttga 20

<210>68

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>68

actcaaaaac atccacaggt ga 22

<210>69

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>69

ggctgtggtg cagtaaccat 20

<210>70

<211>25

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>70

catctacaaa gtaatgcttc ccagt 25

<210>71

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>71

tccctcacgt tattggaagc 20

<210>72

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>72

tccacatcct tcctcaaagg 20

<210>73

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>73

atgatggcca ttttgatgct 20

<210>74

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>74

gctctccacg ttggtaggtc 20

<210>75

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>75

caccaatggc tctgttgtgt 20

<210>76

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>76

taagagctca gcctttattg tgg 23

<210>77

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>77

gctactacat gattggtgag cag 23

<210>78

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>78

ctttaattga ggtcacaggc atc 23

<210>79

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>79

caccgcagca acgttgcagc acattcaaga gatgtgctgc aacgttgctg c 51

<210>80

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>80

aaaagcagca acgttgcagc acatctcttg aatgtgctgc aacgttgctg c 51

<210>81

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA target sequence

<400>81

gcagcaacgt tgcagcaca 19

<210>82

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA hairpin structure

<400>82

gcagcaacgt tgcagcacat tcaagagatg tgctgcaacg ttgctgc 47

<210>83

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>83

ctggaaacag cagccagag 19

<210>84

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>84

cagtgctggc aagacaggta 20

<210>85

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>85

aacttagagg tggggagcag 20

<210>86

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>86

cacaaccatg ccttacttta tc 22

<210>87

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>87

gcaactccta actcccctct gta 23

<210>88

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>88

cagagagcca gagagtgaga gag 23

<210>89

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>89

gcatatacag gagaatgagg tcg 23

<210>90

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>90

ggagtgggaa ccgcagaac 19

<210>91

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>91

gacccccacc ttcaaatcac 20

<210>92

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>92

gctcgtgctc gacaggtgtg ta 22

<210>93

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>93

caggtcatgg ccgggttc 18

<210>94

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>94

gggtcagaaa gtgtctggac tt 22

<210>95

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>95

caacagccca aagattttcc 20

<210>96

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>96

ggtcacgtga taaaatagca caa 23

<210>97

<211>26

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of PCR

<400>97

aacgaattca tggcgtcccc acggga 26

<210>98

<211>28

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of PCR

<400>98

caactcgagc tggtgagcag gcaccgtg 28

<210>99

<211>27

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of PCR

<400>99

caagaattca tgtcgagccc gggcatc 27

<210>100

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of PCR

<400>100

gctgaattca cctcaatgac gaggcagcgg 30

<210>101

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>101

cggtggtctc tcatccttgt 20

<210>102

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>102

gcctcgaggt tatgcttgaa 20

<210>103

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>103

atgcagtcac tcacgctcag 20

<210>104

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>104

tcccgaggac aatccagacg gctttcaaga gaagccgtct ggattgtcct c 51

<210>105

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>105

aaaagaggac aatccagacg gcttctcttg aaagccgtct ggattgtcct c 51

<210>106

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA target sequence

<400>106

gaggacaatc cagacggct 19

<210>107

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hair clip design

<400>107

gaggacaatc cagacggctt tcaagagaag ccgtctggat tgtcctc 47

<210>108

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>108

tcccggagct ctacaacgtg ctgttcaaga gacagcacgt tgtagagctc c 51

<210>109

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>109

aaaaggagct ctacaacgtg ctgtctcttg aacagcacgt tgtagagctc c 51

<210>110

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉SiRNA target sequence

<400>110

ggagctctac aacgtgctg 19

<210>111

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hair clip design

<400>111

ggagctctac aacgtgctgt tcaagagaca gcacgttgta gagctcc 47

<210>112

<211>3503

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(18)..(1427)

<400>112

ctcatcccgg ccccggg atg gcg tcc cca cgg gaa ttg aca cag aac ccc 50

Met Ala Ser Pro Arg Glu Leu Thr Gln Asn Pro

1 5 10

ctg aag aag atc tgg atg cca tac agc aat ggg cgg ccc gct ctg cac 98

Leu Lys Lys Ile Trp Met Pro Tyr Ser Asn Gly Arg Pro Ala Leu His

15 20 25

gct tgc cag cgc ggt gtg tgc atg acc aac tgc cca act ctc att gtc 146

Ala Cys Gln Arg Gly Val Cys Met Thr Asn Cys Pro Thr Leu Ile Val

30 35 40

atg gtg ggc ctg ccc gcc agg ggc aag acc tac atc tcc aag aag ctg 194

Met Val Gly Leu Pro Ala Arg Gly Lys Thr Tyr Ile Ser Lys Lys Leu

45 50 55

act cga tac ctg aac tgg att ggt gtg ccc act cgg gag ttc aat gtt 242

Thr Arg Tyr Leu Asn Trp Ile Gly Val Pro Thr Arg Glu Phe Asn Val

60 65 70 75

ggc cag tat cgc cgg gac gtg gtc aag acc tac aaa tct ttt gaa ttt 290

Gly Gln Tyr Arg Arg Asp Val Val Lys Thr Tyr Lys Ser Phe Glu Phe

80 85 90

ttt ctc ccc gac aat gaa gag ggc ctg aaa atc agg aag cag tgt gcc 338

Phe Leu Pro Asp Asn Glu Glu Gly Leu Lys Ile Arg Lys Gln Cys Ala

95 100 105

ctg gca gcc ctc cgt gac gtc cgg cgg ttc ctt agt gag gag ggg gga 386

Leu Ala Ala Leu Arg Asp Val Arg Arg Phe Leu Ser Glu Glu Gly Gly

110 115 120

cat gtg gcg gtt ttt gat gcc aca aac acc acc cga gaa cgg aga gcg 434

His Val Ala Val Phe Asp Ala Thr Asn Thr Thr Arg Glu Arg Arg Ala

125 130 135

acc atc ttt aat ttt gga gaa cag aat ggc tac aag acc ttt ttt gtc 482

Thr Ile Phe Asn Phe Gly Glu Gln Asn Gly Tyr Lys Thr Phe Phe Val

140 145 150 155

gag tcc atc tgt gtg gat cct gag gtc ata gct gcc aac atc gtg caa 530

Glu Ser Ile Cys Val Asp Pro Glu Val Ile Ala Ala Asn Ile Val Gln

160 165 170

gtg aaa ctg ggc agc cct gac tat gtc aac cgc gac agt gat gag gct 578

Val Lys Leu Gly Ser Pro Asp Tyr Val Asn Arg Asp Ser Asp Glu Ala

175 180 185

acg gag gac ttc atg agg cgc att gag tgc tat gag aac tcc tac gag 626

Thr Glu Asp Phe Met Arg Arg Ile Glu Cys Tyr Glu Asn Ser Tyr Glu

190 195 200

tcg cta gat gag gac ctg gat agg gac ctg tcc tat atc aag atc atg 674

Ser Leu Asp Glu Asp Leu Asp Arg Asp Leu Ser Tyr Ile Lys Ile Met

205 210 215

gat gtg ggc cag agc tac gtg gtg aac cgt gtg gct gac cac atc cag 722

Asp Val Gly Gln Ser Tyr Val Val Asn Arg Val Ala Asp His Ile Gln

220 225 230 235

agc cgc atc gta tat tac ctc atg aac atc cac gtg acc ccc cgc tcc 770

Ser Arg Ile Val Tyr Tyr Leu Met Asn Ile His Val Thr Pro Arg Ser

240 245 250

atc tac ctc tgc cgg cac ggg gag agc gag ctc aac ctc aag ggc cgg 818

Ile Tyr Leu Cys Arg His Gly Glu Ser Glu Leu Asn Leu Lys Gly Arg

255 260 265

att ggc ggg gac cca gga ctg tcc cct cgg ggc agg gag ttt gcc aag 866

Ile Gly Gly Asp Pro Gly Leu Ser Pro Arg Gly Arg Glu Phe Ala Lys

270 275 280

agt cta gcc cag ttc atc agt gac caa aat atc aag gat ctg aag gtc 914

Ser Leu Ala Gln Phe Ile Ser Asp Gln Asn Ile Lys Asp Leu Lys Val

285 290 295

tgg aca agc cag atg aag agg aca atc cag acg gct gag gca ctg ggt 962

Trp Thr Ser Gln Met Lys Arg Thr Ile Gln Thr Ala Glu Ala Leu Gly

300 305 310 315

gtg ccc tat gaa cag tgg aag gtc ctc aac gag atc gat gcg ggc gtc 1010

Val Pro Tyr Glu Gln Trp Lys Val Leu Asn Glu Ile Asp Ala Gly Val

320 325 330

tgt gag gaa atg acc tac gag gaa att cag gat aat tat cca ctg gag 1058

Cys Glu Glu Met Thr Tyr Glu Glu Ile Gln Asp Asn Tyr Pro Leu Glu

335 340 345

ttc gcc ctg cgg gac cag gac aag tac cgg tac cgg tac cct aaa ggg 1106

Phe Ala Leu Arg Asp Gln Asp Lys Tyr Arg Tyr Arg Tyr Pro Lys Gly

350 355 360

gag tcc tac gag gac ctg gtc cag aga ctg gag cct gtc atc atg gag 1154

Glu Ser Tyr Glu Asp Leu Val Gln Arg Leu Glu Pro Val Ile Met Glu

365 370 375

ctg gag agg caa gag aat gtg ctg gtc atc tgc cac cag gct gtg atg 1202

Leu Glu Arg Gln Glu Asn Val Leu Val Ile Cys His Gln Ala Val Met

380 385 390 395

cgc tgc ctg ctg gcc tac ttc ctc gac aag gca gca gaa cag ctg ccc 1250

Arg Cys Leu Leu Ala Tyr Phe Leu Asp Lys Ala Ala Glu Gln Leu Pro

400 405 410

tac ctc aag tgt ccg ctg cac aca gtc ctg aag ctg act cct gtg gca 1298

Tyr Leu Lys Cys Pro Leu His Thr Val Leu Lys Leu Thr Pro Val Ala

415 420 425

tat ggt tgt aaa gtg gag tcc ata ttc ctg aac gtg gct gct gtg aac 1346

Tyr Gly Cys Lys Val Glu Ser Ile Phe Leu Asn Val Ala Ala Val Asn

430 435 440

acg cac cgg gac agg cct cag aac gtg gac atc tca aga cct cca gag 1394

Thr His Arg Asp Arg Pro Gln Asn Val Asp Ile Ser Arg Pro Pro Glu

445 450 455

gaa gcc ctt gtc acg gtg cct gct cac cag tga ccatgttcat ccactgtgac 1447

Glu Ala Leu Val Thr Val Pro Ala His Gln

460 465

cactaggcag gcactgctct ctgcagaggg ggtcattcca ggccctccag tgtgtgtgat 1507

agtcaccatg ccatgcaggg atattcttga agccacacat ggctggcgga acccagagcc 1567

cccaccccag cccacctggc tctttgttga cagtcggcga caaggttgtg cgtggctcct 1627

gacctgctgc taagagtcac ttgaccagac tgcatctgca tgggctgcgc ggaggttgcc 1687

cagccccagt ttcttccggc gcagctctta ggtgttcact ctcgccagct cagttggctt 1747

tgtgaagtgt gaaaccctac aatgtgaaag gaaagtgctt gctgtgatgt tcctactgtg 1807

gcccagctgc ccagcatgga cctggtgact ctccacaggg cctctaccat cctctctgtg 1867

gccacttcct gagccagagg ccaggtcttc atggggccct gagcttctgc tgcctctggt 1927

gagagggaga gcccttccca tccttaccca ccaggaacta gagccccaac cacagcagat 1987

gcttcctggg cagccactgg ccaggccgtt gtatccatgt cacccttagt tgtgggcatt 2047

catgaaagca atgcgcttgc ttcagcacat tgggatgcac agaaacgtga gggcaggggg 2107

gcagttgtgg tcgcccctcc ccagttgtgc atgtacccgg ctacttaggt atgcttaggg 2167

gagctggtgg ccaggggtgc cccttcaggc ctgctctcac agtgaaacca cttcaagaca 2227

gggcaggaag ttaccttcct ggtgcacaca ccgaagtgct cgagggagag gctgccgccc 2287

cctctcccct tccctggctg tggacactgg cagccaatgg ggaggcggcc tctcccctgt 2347

tgctgcccgt gtccacacag gaatgtacac tggtggcaga aacatgcatt tgccataaat 2407

aattcagaaa cacttgtatg gtcctgtaaa acttctctgg tgtttgttga cttctgcctg 2467

tgggtctgag gaagggagaa ggcagaaccc ataggcagtc cctgggcaag ggaggatttt 2527

cccggccgca gcacagcctt ccattcatgt gcacgtgagt ctggtgaaaa tggccttgtg 2587

aagtccagtg tctgtcatgc ccctgagttc ttttggtccc agagaggcag cctgggcagt 2647

ctgtttacag ctggcaaaca gactggctgg caagttgtgg ggctgggtca gaaagtgtct 2707

ggacttgtaa tcgggtgccc tgctgtccac aggtggactc tggggaagcc aggcccctgg 2767

atcccaagtc ctttgtgtcc ggagcctttc ctggcctgtg cctggagggt agcacatctt 2827

tccagcctcc cagggctttg cagcctgcag atgccacctg tccctgagag aagttagcgt 2887

ccctggccct ggccggtggt ctctcatcct tgtgtgctgc tctgctaaga gatgtccaag 2947

gcggagccgg ggcaagatcc ttccagactc atctgtcaga gccccaagcc ctttagaccc 3007

agagcccaag gaccatgcct ttgggacatt aggactgcag cctttgcttc tgtgtatttt 3067

ggagttttgg tgacttttgt cacctggaca cactcatttg ttagccatag tgggttccct 3127

tggtcagcaa cagtgcatgt acctctggat gtcatctgag gtgagaccac cgaggccttt 3187

tctctctgtg tacagagggg agttaggagt tgctggactg gatgcattac gaggactggg 3247

gacagggtag agggacatcc agggatcagg gcatgagtgg gggcaacccc ccggcctctg 3307

ccctggcatg gtctccgcat gggctgaggt gtagctgatt ggctgccaca tttcggccat 3367

gctggctggc gtgcccatgt tgcagatatt ttcccgagtt ccccagaatg gatggtattg 3427

aatctcagcc acatgcaaca ctgtgtccag cattctttgc aataaatact ttttaaaaaa 3487

taaaaaaaaa aaaaaa 3503

<210>113

<211>469

<212>PRT

＜213〉people (Homo sapiens)

<400>113

Met Ala Ser Pro Arg Glu Leu Thr Gln Asn Pro Leu Lys Lys Ile Trp

1 5 10 15

Met Pro Tyr Ser Asn Gly Arg Pro Ala Leu His Ala Cys Gln Arg Gly

20 25 30

Val Cys Met Thr Asn Cys Pro Thr Leu Ile Val Met Val Gly Leu Pro

35 40 45

Ala Arg Gly Lys Thr Tyr Ile Ser Lys Lys Leu Thr Arg Tyr Leu Asn

50 55 60

Trp Ile Gly Val Pro Thr Arg Glu Phe Asn Val Gly Gln Tyr Arg Arg

65 70 75 80

Asp Val Val Lys Thr Tyr Lys Ser Phe Glu Phe Phe Leu Pro Asp Asn

85 90 95

Glu Glu Gly Leu Lys Ile Arg Lys Gln Cys Ala Leu Ala Ala Leu Arg

100 105 110

Asp Val Arg Arg Phe Leu Ser Glu Glu Gly Gly His Val Ala Val Phe

115 120 125

Asp Ala Thr Asn Thr Thr Arg Glu Arg Arg Ala Thr Ile Phe Asn Phe

130 135 140

Gly Glu Gln Asn Gly Tyr Lys Thr Phe Phe Val Glu Ser Ile Cys Val

145 150 155 160

Asp Pro Glu Val Ile Ala Ala Asn Ile Val Gln Val Lys Leu Gly Ser

165 170 175

Pro Asp Tyr Val Asn Arg Asp Ser Asp Glu Ala Thr Glu Asp Phe Met

180 185 190

Arg Arg Ile Glu Cys Tyr Glu Asn Ser Tyr Glu Ser Leu Asp Glu Asp

195 200 205

Leu Asp Arg Asp Leu Ser Tyr Ile Lys Ile Met Asp Val Gly Gln Ser

210 215 220

Tyr Val Val Asn Arg Val Ala Asp His Ile Gln Ser Arg Ile Val Tyr

225 230 235 240

Tyr Leu Met Asn Ile His Val Thr Pro Arg Ser Ile Tyr Leu Cys Arg

245 250 255

His Gly Glu Ser Glu Leu Asn Leu Lys Gly Arg Ile Gly Gly Asp Pro

260 265 270

Gly Leu Ser Pro Arg Gly Arg Glu Phe Ala Lys Ser Leu Ala Gln Phe

275 280 285

Ile Ser Asp Gln Asn Ile Lys Asp Leu Lys Val Trp Thr Ser Gln Met

290 295 300

Lys Arg Thr Ile Gln Thr Ala Glu Ala Leu Gly Val Pro Tyr Glu Gln

305 310 315 320

Trp Lys Val Leu Asn Glu Ile Asp Ala Gly Val Cys Glu Glu Met Thr

325 330 335

Tyr Glu Glu Ile Gln Asp Asn Tyr Pro Leu Glu Phe Ala Leu Arg Asp

340 345 350

Gln Asp Lys Tyr Arg Tyr Arg Tyr Pro Lys Gly Glu Ser Tyr Glu Asp

355 360 365

Leu Val Gln Arg Leu Glu Pro Val Ile Met Glu Leu Glu Arg Gln Glu

370 375 380

Asn Val Leu Val Ile Cys His Gln Ala Val Met Arg Cys Leu Leu Ala

385 390 395 400

Tyr Phe Leu Asp Lys Ala Ala Glu Gln Leu Pro Tyr Leu Lys Cys Pro

405 410 415

Leu His Thr Val Leu Lys Leu Thr Pro Val Ala Tyr Gly Cys Lys Val

420 425 430

Glu Ser Ile Phe Leu Asn Val Ala Ala Val Asn Thr His Arg Asp Arg

435 440 445

Pro Gln Asn Val Asp Ile Ser Arg Pro Pro Glu Glu Ala Leu Val Thr

450 455 460

Val Pro Ala His Gln

465

<210>114

<211>2539

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(500)..(754)

<400>114

ccgagaacct gcgcaagctg cgcagccttg acctctcgtg gtgcccacgc atcaccgaca 60

tggcgctgga gtacgtggcc tgcgacctgc accgcctaga ggagctcgtg ctcgacaggt 120

gcgcgccccc gggccgcgcc gggcggggct gggccgggcg gggctgcacg gggcggggcg 180

gggcggggct gcacggggcc gggggcgggg gcttccggcg gggctggggc tgtggcgcgg 240

cgggcagagc cctcggagcc tggggagtgg gaaccgcaga accgcagcac gggttggctg 300

gacccccgtc cttcccggtg ggtgggagcg aggaggggcc agcggctgct ccgggtgggg 360

caggaaggga ttcacgccag gggcactccc aggaagacga gggatggccg gggccaggtg 420

atttgaaggt gggggtcccc tctggtgcaa ccacaggtgt gtacgcatca cggacactgg 480

cctcagctat ctgtccacc atg tcg tcc ctc cgc agc ctc tac ctg cga tgg 532

Met Ser Ser Leu Arg Ser Leu Tyr Leu Arg Trp

1 5 10

tgc tgc cag gtg caa gac ttc ggg ctg aag cac ctc ctg gcc ctg ggg 580

Cys Cys Gln Val Gln Asp Phe Gly Leu Lys His Leu Leu Ala Leu Gly

15 20 25

agt ttg cgc ctc ctg tct ctg gca ggc tgc ccg ctg ctc acc acc acc 628

Ser Leu Arg Leu Leu Ser Leu Ala Gly Cys Pro Leu Leu Thr Thr Thr

30 35 40

ggg ctg tcg ggc ctg gtg cag ctg cag gag ctg gag gag ctg gag ctg 676

Gly Leu Ser Gly Leu Val Gln Leu Gln Glu Leu Glu Glu Leu Glu Leu

45 50 55

acc aac tgc ccc ggg gcc acc ccc gag ctc ttc aag tat ttc tcg cag 724

Thr Asn Cys Pro Gly Ala Thr Pro Glu Leu Phe Lys Tyr Phe Ser Gln

60 65 70 75

cac ctg ccc cgc tgc ctc gtc att gag tag cgcgaggccc ccgccccggt 774

His Leu Pro Arg Cys Leu Val Ile Glu

80

cgcgggaacc cggccatgac ctgggcgggg gcgcggggcg ccgccgagcc ccctcttccc 834

gccttgcgct cgggggagcc cccgcgcccc cggcccagcg cgggaggcgg ggcgagccga 894

gggaaagccc ctccccgacc ttcggtccct ccgccctccc agccccgccc cgggcagggg 954

ggcggcgggt gggcccgccc cacgcacgca cgcacactcg gggactttgt gcatgcccct 1014

cgtgcccgca ctgcacgccg ccctccgcca cgcccacagc cacagccgcc gccatcactc 1074

gctcgccctc ccgcttgggg ggcggggctc ggtccttggg ggggctttga gctctccaga 1134

ctgtgccctt accgccttcc ccgccacacc cgctctgtct tcccactgtc ccccccatcc 1194

cgggcagggc ccagtgggat tgagggggct gggtccccca ggacacgggc ccagaagagc 1254

cccacgggct tcctgcatct tccaccgcac catacctgga gccctccgag gggtgtcagg 1314

ggaaacaggc caccgccaaa gccatggccc gccgccgaga gcccaagccc cacccgcacc 1374

tcctcaccca tccagcctga cccacgcggc ctctcctcct ccttgccgct gtgtggggca 1434

gtcccctgtc cgccccaaaa cccggccttg gtccctggcc aggctgagag aattgggcag 1494

ggagagggcg gaagggctgg cgatcgcttg gagtcattaa cgtgatccca gctgactccg 1554

gtcggcctca acccaggggt ggcgcaggca ccttgcaagc ctcgagctgt agccaccctc 1614

aggcctggga agaggcctgg gccgacctca cacctcagcc cttgcacccg gccgggctca 1674

gttcaggcct gggcaccgag cttcaccctg ggtgggtctc ctcaggtgga gtctgcagag 1734

tggacccagc caagggtcag ggtcagcact gggtcagcga ctccaatctt ccagtggcca 1794

gcacacccta gacaccccga ggagggaggg ctcctttcta gcctgccccc ccacccccac 1854

ttcacccctc cccagcttcc caaacttctg tctgcccaaa tgggctctga ccgtgctctg 1914

tcggcccgag acatttggaa gtcctggggg atgctggcaa atctcagctg ttgctgagga 1974

gggggctggg accccttccc atcccaacct tgagccccag gagataccgc gcccacaccc 2034

aatcttggga cactccctat ctggttggaa gagagtaacc agtttccaga gagccagaga 2094

gtgagagaga gaaagagagt gagagagaga gagaaagaga gagagagatg ctgttgaatc 2154

agaaacagat caacagccca aagattttcc tgtccctgga gtgccagccc caggaagctc 2214

cagggctgag tggtcaggag ccagtttctc cagcccctcc tccccacaac ccctagtggg 2274

gaggggcagc tgtccatttg cccaaagtat taatgcaact gaagctgtga tatttccaac 2334

gactgtagga ggaaaaatta aggggagaga ggaaaacaaa accaaccaac ccctaaaatc 2394

attttcttat tgtacataac gacctcattc tcctgtatat gcggaagata taaccttata 2454

tttggtaagt gtttcttgtg ctattttatc acgtgacctg tttataaaaa tatatattaa 2514

aaaagttgta aaaaaaaaaa aaaaa 2539

<210>115

<211>84

<212>PRT

＜213〉people (Homo sapiens)

<400>115

Met Ser Ser Leu Arg Ser Leu Tyr Leu Arg Trp Cys Cys Gln Val Gln

1 5 10 15

Asp Phe Gly Leu Lys His Leu Leu Ala Leu Gly Ser Leu Arg Leu Leu

20 25 30

Ser Leu Ala Gly Cys Pro Leu Leu Thr Thr Thr Gly Leu Ser Gly Leu

35 40 45

Val Gln Leu Gln Glu Leu Glu Glu Leu Glu Leu Thr Asn Cys Pro Gly

50 55 60

Ala Thr Pro Glu Leu Phe Lys Tyr Phe Ser Gln His Leu Pro Arg Cys

65 70 75 80

Leu Val Ile Glu

<210>116

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial sequence synthesized primer of RT-PCR

<400>116

cttccagact catctgtcag agc 23

<210>117

<211>3427

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(204)..(1643)

<400>117

aggggaggag gaggcgcccg cctggttccc tgcaaagcgg ccttatttat ctgggcacag 60

cctcagcctc cccggtggga ggcttggggc ggccgatcct ctcccaccgg ggagctcctt 120

tccgtgcgct ccccatgacg ggaggtcgcc cggcggacag cgtggcatga gccaggagcc 180

cgggccgaga gcgtgccagg aag atg tcg agc ccg ggc atc gac ggc gac ccc 233

Met Ser Ser Pro Gly Ile Asp Gly Asp Pro

1 5 10

aag cct cca tgc ttg cct cga aac ggt ctg gtg aag ctg ccg ggc cag 281

Lys Pro Pro Cys Leu Pro Arg Asn Gly Leu Val Lys Leu Pro Gly Gln

15 20 25

ccc aac ggc ctg ggt gcg gcc agc atc acc aag ggc acg cca gcc acc 329

Pro Asn Gly Leu Gly Ala Ala Ser Ile Thr Lys Gly Thr Pro Ala Thr

30 35 40

aag aac cgc ccc tgc cag cca cca ccc cca ccc acc ctc cca cca cca 377

Lys Asn Arg Pro Cys Gln Pro Pro Pro Pro Pro Thr Leu Pro Pro Pro

45 50 55

agc ctg gct gct cca ctg tcc cgg gct gcc ctg gct ggg ggc ccg tgc 425

Ser Leu Ala Ala Pro Leu Ser Arg Ala Ala Leu Ala Gly Gly Pro Cys

60 65 70

acc ccg gca ggt gga cca gcc tca gcc ttg gca cct ggg cac cca gcg 473

Thr Pro Ala Gly Gly Pro Ala Ser Ala Leu Ala Pro Gly His Pro Ala

75 80 85 90

gag cgg ccg ccg ctg gcc acg gac gag aag atc ctc aat ggg ctc ttc 521

Glu Arg Pro Pro Leu Ala Thr Asp Glu Lys Ile Leu Asn Gly Leu Phe

95 100 105

tgg tat ttc tcg gcc tgc gag aag tgt gtg ctg gcc cag gtg tgc aag 569

Trp Tyr Phe Ser Ala Cys Glu Lys Cys Val Leu Ala Gln Val Cys Lys

110 115 120

gcc tgg cgg cgc gtg ctg tac cag ccc aag ttc tgg gca ggc ctc acg 617

Ala Trp Arg Arg Val Leu Tyr Gln Pro Lys Phe Trp Ala Gly Leu Thr

125 130 135

ccg gtg ctg cat gcc aag gag ctc tac aac gtg ctg cct ggt ggc gag 665

Pro Val Leu His Ala Lys Glu Leu Tyr Asn Val Leu Pro Gly Gly Glu

140 145 150

aag gag ttc gtg aac ctg cag ggt ttt gcc gcc aga ggc ttc gag ggc 713

Lys Glu Phe Val Asn Leu Gln Gly Phe Ala Ala Arg Gly Phe Glu Gly

155 160 165 170

ttc tgc ctg gtt ggc gtc tcc gac ctg gac atc tgt gag ttc att gac 761

Phe Cys Leu Val Gly Val Ser Asp Leu Asp Ile Cys Glu Phe Ile Asp

175 180 185

aac tat gcg ctc tcc aag aag ggt gtc aaa gcc atg agc ctc aag cgc 809

Asn Tyr Ala Leu Ser Lys Lys Gly Val Lys Ala Met Ser Leu Lys Arg

190 195 200

tcc acc atc acg gac gca ggc ctc gag gtt atg ctt gaa cag atg cag 857

Ser Thr Ile Thr Asp Ala Gly Leu Glu Val Met Leu Glu Gln Met Gln

205 210 215

ggc gtg gtg cgt ctg gag ctg tcg ggc tgc aac gac ttc acc gag gcc 905

Gly Val Val Arg Leu Glu Leu Ser Gly Cys Asn Asp Phe Thr Glu Ala

220 225 230

ggg ctg tgg tcc agc ctg agc gcg cgc atc acc tcg ctg agc gtg agt 953

Gly Leu Trp Ser Ser Leu Ser Ala Arg Ile Thr Ser Leu Ser Val Ser

235 240 245 250

gac tgc atc aac gtg gcc gac gac gcc atc gcg gcc atc tcg cag ctg 1001

Asp Cys Ile Asn Val Ala Asp Asp Ala Ile Ala Ala Ile Ser Gln Leu

255 260 265

ctg ccc aac ctg gcg gag ctg agc ctg cag gcc tac cac gtg acg gac 1049

Leu Pro Asn Leu Ala Glu Leu Ser Leu Gln Ala Tyr His Val Thr Asp

270 275 280

acg gcg ctg gcc tac ttc acg gcg cgc cag ggc cac agc acg cac acg 1097

Thr Ala Leu Ala Tyr Phe Thr Ala Arg Gln Gly His Ser Thr His Thr

285 290 295

ctg cgc ctg ctc tcc tgc tgg gag atc acc aac cac ggc gtg gtc aac 1145

Leu Arg Leu Leu Ser Cys Trp Glu Ile Thr Asn His Gly Val Val Asn

300 305 310

gtg gtg cac agc ctg ccc aac ctc acc gcg ctc agc ctc tcg ggc tgc 1193

Val Val His Ser Leu Pro Asn Leu Thr Ala Leu Ser Leu Ser Gly Cys

315 320 325 330

tcc aag gtc acc gac gac ggc gtg gag ctc gtg gcc gag aac ctg cgc 1241

Ser Lys Val Thr Asp Asp Gly Val Glu Leu Val Ala Glu Asn Leu Arg

335 340 345

aag ctg cgc agc ctt gac ctc tcg tgg tgc cca cgc atc acc gac atg 1289

Lys Leu Arg Ser Leu Asp Leu Ser Trp Cys Pro Arg Ile Thr Asp Met

350 355 360

gcg ctg gag tac gtg gcc tgc gac ctg cac cgc cta gag gag ctc gtg 1337

Ala Leu Glu Tyr Val Ala Cys Asp Leu His Arg Leu Glu Glu Leu Val

365 370 375

ctc gac agg tgt gta cgc atc acg gac act ggc ctc agc tat ctg tcc 1385

Leu Asp Arg Cys Val Arg Ile Thr Asp Thr Gly Leu Ser Tyr Leu Ser

380 385 390

acc atg tcg tcc ctc cgc agc ctc tac ctg cga tgg tgc tgc cag gtg 1433

Thr Met Ser Ser Leu Arg Ser Leu Tyr Leu Arg Trp Cys Cys Gln Val

395 400 405 410

caa gac ttc ggg ctg aag cac ctc ctg gcc ctg ggg agt ttg cgc ctc 1481

Gln Asp Phe Gly Leu Lys His Leu Leu Ala Leu Gly Ser Leu Arg Leu

415 420 425

ctg tct ccg gca ggc tgc ccg ctg ctc acc acc acc ggg ctg tcg ggc 1529

Leu Ser Pro Ala Gly Cys Pro Leu Leu Thr Thr Thr Gly Leu Ser Gly

430 435 440

ctg gtg cag ctg cag gag ctg gag gag ctg gag ctg acc aac tgc ccc 1577

Leu Val Gln Leu Gln Glu Leu Glu Glu Leu Glu Leu Thr Asn Cys Pro

445 450 455

ggg gcc acc ccc gag ctc ttc aag tat ttc tcg cag cac ctg ccc cgc 1625

Gly Ala Thr Pro Glu Leu Phe Lys Tyr Phe Ser Gln His Leu Pro Arg

460 465 470

tgc ctc gtc att gag tag cgcgaggccc ccgccccggt cgcgggaacc 1673

Cys Leu Val Ile Glu

475

cggccatgac ctgggcgggg gcgcggggcg ccgccgagcc ccctcttccc gccttgcgct 1733

cgggggagcc cccgcgcccc cggcccagcg cgggagacgg ggcgagccga gggaaagccc 1793

ctccccgacc ttcggtccct ccgccctccc agccccgccc cgggcagggg ggcggcgggt 1853

gggcccgccc cacgcacgca cgcacactcg gggactttgt gcatgcccct cgtgcccgca 1913

ctgcacgccg ccctccgcca cgccacagcc acagccgccg ccatcactcg ctcgccctcc 1973

cgcttggggg gcggggctcg gtccttgggg gggctttgag ctctccagac tgtgccctta 2033

ccgccttccc cgccacaccc gctctgtctt cccactgtcc ccccaatccc gggcagggcc 2093

cagtgggatt gagggggctg ggtcccccag gacacgggcc cagaagagcc ccacgggctt 2153

cctgcatctt ccaccgcacc atacctggag ccctccgagg ggtgtcaggg gaaacaggcc 2213

accgccaaag ccatggcccg ccgccgagag cccaggcccc acccgcacct cctcacccat 2273

ccagcctgac ccacgcggcc tctcctcctc cttgccgctg tgtggggcag tcccctgtcc 2333

gccccaaaac ccggccttgg tccctggcca ggctgagaga attgggcagg gagagggcgg 2393

aagggctggc gatcgcttgg agtcattaac gtgatccaag ctgactccgg tcggcctcaa 2453

cccaggggtg gcgcaggcac cttgcaagcc tcgagctgta gccaccctca ggcctgggaa 2513

gaggcctggg ccgacctcac acctcagccc ttgcacccgg ccgggctcag ttcaggcctg 2573

ggcaccgagc ttcaccctgg gtgggtctcc tcaggtggag tctgcagagt ggacccagcc 2633

aagggtcagg gtcagcactg ggtcagcgac tccaatcttc cagtggccag cacaccctag 2693

acaccccgag gagggagggc tcctttctag cctgcccccc cacccccact tcacccctcc 2753

ccagcttccc aaacttctgt ctgcccaaat gggctctgac cgtgctctgt cggcccgaga 2813

catttggaag tcctggggga tgctggcaaa tctcagctgt tgctgaggag ggggctggga 2873

ccccttccca tcccaacctt gagccccagg agataccgcg cccacaccca atcttgggac 2933

actccctatc tggttggaag agagtaacca gtttccagag agccagagag tgagagagag 2993

aaagagagtg agagagagag agaaagagag agagagatgc tgttgaatca gaaacagatc 3053

aacagcccaa agattttcct gtccctggag tgccagcccc aggaagctcc agggctgagt 3113

ggtcaggagc cagtttctcc agcccctcct ccccacaacc cctagtgggg aggggcagct 3173

gtccatttgc ccaaagtatt aatgcaactg aagctgtgat atttccaacg actgtaggag 3233

gaaaaattaa ggggagagag gaaaacaaaa ccaaccaacc cctaaaatca ttttcttatt 3293

gtacataacg acctcattct cctgtatatg cggaagatat aaccttatat ttggtaagtg 3353

tttcttgtgc tattttatca cgtgacctgt ttataaaaat atatattaaa aaagttgtaa 3413

aaaaaaaaaa aaaa 3427

<210>118

<211>479

<212>PRT

＜213〉people (Homo sapiens)

<400>118

Met Ser Ser Pro Gly Ile Asp Gly Asp Pro Lys Pro Pro Cys Leu Pro

1 5 10 15

Arg Asn Gly Leu Val Lys Leu Pro Gly Gln Pro Asn Gly Leu Gly Ala

20 25 30

Ala Ser Ile Thr Lys Gly Thr Pro Ala Thr Lys Asn Arg Pro Cys Gln

35 40 45

Pro Pro Pro Pro Pro Thr Leu Pro Pro Pro Ser Leu Ala Ala Pro Leu

50 55 60

Ser Arg Ala Ala Leu Ala Gly Gly Pro Cys Thr Pro Ala 6ly Gly Pro

65 70 75 80

Ala Ser Ala Leu Ala Pro Gly His Pro Ala Glu Arg Pro Pro Leu Ala

85 90 95

Thr Asp Glu Lys Ile Leu Asn Gly Leu Phe Trp Tyr Phe Ser Ala Cys

100 105 110

Glu Lys Cys Val Leu Ala Gln Val Cys Lys Ala Trp Arg Arg Val Leu

115 120 125

Tyr Gln Pro Lys Phe Trp Ala Gly Leu Thr Pro Val Leu His Ala Lys

130 135 140

Glu Leu Tyr Asn Val Leu Pro Gly Gly Glu Lys Glu Phe Val Asn Leu

145 150 155 160

Gln Gly Phe Ala Ala Arg Gly Phe Glu Gly Phe Cys Leu Val Gly Val

165 170 175

Ser Asp Leu Asp Ile Cys Glu Phe Ile Asp Asn Tyr Ala Leu Ser Lys

180 185 190

Lys Gly Val Lys Ala Met Ser Leu Lys Arg Ser Thr Ile Thr Asp Ala

195 200 205

Gly Leu Glu Val Met Leu Glu Gln Met Gln Gly Val Val Arg Leu Glu

210 215 220

Leu Ser Gly Cys Asn Asp Phe Thr Glu Ala Gly Leu Trp Ser Ser Leu

225 230 235 240

Ser Ala Arg Ile Thr Ser Leu Ser Val Ser Asp Cys Ile Asn Val Ala

245 250 255

Asp Asp Ala Ile Ala Ala Ile Ser Gln Leu Leu Pro Asn Leu Ala Glu

260 265 270

Leu Ser Leu Gln Ala Tyr His Val Thr Asp Thr Ala Leu Ala Tyr Phe

275 280 285

Thr Ala Arg Gln Gly His Ser Thr His Thr Leu Arg Leu Leu Ser Cys

290 295 300

Trp Glu Ile Thr Asn His Gly Val Val Asn Val Val His Ser Leu Pro

305 310 315 320

Asn Leu Thr Ala Leu Ser Leu Ser Gly Cys Ser Lys Val Thr Asp Asp

325 330 335

Gly Val Glu Leu Val Ala Glu Asn Leu Arg Lys Leu Arg Ser Leu Asp

340 345 350

Leu Ser Trp Cys Pro Arg Ile Thr Asp Met Ala Leu Glu Tyr Val Ala

355 360 365

Cys Asp Leu His Arg Leu Glu Glu Leu Val Leu Asp Arg Cys Val Arg

370 375 380

Ile Thr Asp Thr Gly Leu Ser Tyr Leu Ser Thr Met Ser Ser Leu Arg

385 390 395 400

Ser Leu Tyr Leu Arg Trp Cys Cys Gln Val Gln Asp Phe Gly Leu Lys

405 410 415

His Leu Leu Ala Leu Gly Ser Leu Arg Leu Leu Ser Pro Ala Gly Cys

420 425 430

Pro Leu Leu Thr Thr Thr Gly Leu Ser Gly Leu Val Gln Leu Gln Glu

435 440 445

Leu Glu Glu Leu Glu Leu Thr Asn Cys Pro Gly Ala Thr Pro Glu Leu

450 455 460

Phe Lys Tyr Phe Ser Gln His Leu Pro Arg Cys Leu Val Ile Glu

465 470 475

Claims (54)

1. tendentious method of diagnosing the RCC among the experimenter or RCC taking place, this method comprises the RCC Expression of Related Genes level in the biological sample that is derived from the patient of measuring, wherein, the expression level of described gene in described sample this experimenter of expression that rises compared with the normal control level or descend suffers from RCC or the danger of RCC exist to take place.

2. the process of claim 1 wherein that described RCC genes involved is selected from RCC 1-251, and compared with the normal control level, the expression level in the described sample rises and represents that described experimenter suffers from RCC or has the danger that RCC takes place.

3. the process of claim 1 wherein, compare the expression level height at least 10% in the described sample with described normal control level.

4. the process of claim 1 wherein that described RCC genes involved is selected from RCC 252-972, and compared with the normal control level, the expression level in the described sample descends and represents that described experimenter suffers from RCC or has the danger that RCC takes place.

5. the method for claim 4 wherein, is compared with described normal control level, the expression level in the described sample low at least 10%.

6. the method for claim 1, described method further comprise a plurality of RCC Expression of Related Genes levels of measuring.

7. the method for claim 1, it adopts the method that is selected from down group to measure the expression of gene level:

(a) mRNA of detection RCC genes involved,

(b) detect by the related gene coded albumen of RCC and

(c) detect by the related gene coded proteic biological activity of RCC.

8. the process of claim 1 wherein that described hybridization step carries out on the DNA array.

9. the process of claim 1 wherein that the described patient's of being derived from biological sample comprises epithelial cell.

10. the process of claim 1 wherein that the described patient's of being derived from biological sample comprises the renal cell carcinoma cell.

11. the method for claim 7, the wherein said patient's of being derived from biological sample comprises the epithelial cell from renal cell carcinoma.

12. RCC reference table expression patterns, it comprises that the genetic expression of RCC genes involved more than 2 kinds that is selected from RCC 1-972 is graphic.

13. RCC reference table expression patterns, it comprises that the genetic expression of RCC genes involved more than 2 kinds that is selected from RCC 1-251 is graphic.

14. RCC reference table expression patterns, it comprises that the genetic expression of RCC genes involved more than 2 kinds that is selected from RCC 252-972 is graphic.

15. a screening is used for the treatment of or prevents the method for the compound of renal cell carcinoma, said method comprising the steps of:

A) test-compound is contacted with polypeptide, described polypeptide is by the polynucleotide encoding that is selected from RCC 1-972;

B) detect the activity that combine between polypeptide and the test-compound; With

C) select and polypeptide bonded test-compound.

16. a screening is used for the treatment of or prevents the method for the compound of renal cell carcinoma, said method comprising the steps of:

A) make candidate compound and the cells contacting of expressing one or more marker gene, wherein said one or more marker gene are selected from RCC 1-972; With

B) select such candidate compound: compared with the control, described candidate compound reduces the expression level of the one or more marker gene that are selected from RCC1-251, perhaps improves the expression level of the one or more marker gene that are selected from RCC 252-972.

17. the method for claim 16, wherein, described cell comprises the renal cell carcinoma cell.

18. a screening is used for the treatment of or prevents the method for the compound of renal cell carcinoma, said method comprising the steps of:

A) test-compound is contacted with polypeptide, described polypeptide is by the polynucleotide encoding that is selected from RCC 1-972;

B) biological activity of the polypeptide of detection step a); With

C) select such test-compound: the biological activity of this polypeptide that detects when not having test-compound is compared, and described test-compound suppresses the biological activity by the polypeptide of the polynucleotide encoding that is selected from RCC 1-251; Perhaps, the biological activity of this polypeptide that detects when not having test-compound is compared, and described test-compound strengthens the biological activity by the polypeptide of the polynucleotide encoding that is selected from RCC 252-972.

19. a screening is used for the treatment of or prevents the method for the compound of renal cell carcinoma, said method comprising the steps of:

A) make candidate compound and the cells contacting that has wherein imported carrier, wherein said carrier contains the transcription regulatory region of one or more marker gene and the reporter gene of expressing under the control of this transcription regulatory region, wherein said one or more marker gene are selected from RCC 1-972;

B) expression or the activity of the described reporter gene of measurement; With

C) select such candidate compound: compare with the expression level or the activity of the described reporter gene that when not having test-compound, detects, when described marker gene is when being selected from the rise marker gene of gene RCC 1-251, described candidate compound reduces the expression or the activity level of described reporter gene, perhaps when described marker gene be that described candidate compound strengthens the expression or the activity level of described reporter gene when being selected from the downward modulation marker gene of gene RCC 252-972.

20. a test kit contains detection reagent, described detection reagent and the nucleotide sequence more than 2 kinds that (a) is selected from RCC 1-972 are perhaps combined by its encoded polypeptides with (b).

21. an array, it contains and is selected from a kind of RCC 1-972 or multiple nucleotide sequence bonded nucleic acid more than 2 kinds.

22. the method for the treatment of or preventing renal cell carcinoma among the experimenter, it comprises uses the antisense composition to described experimenter, and described antisense composition comprises and the encoding sequence complementary nucleotide sequence that is selected from RCC 1-251.

23. the method for the treatment of or preventing renal cell carcinoma among the experimenter, it comprises uses the siRNA composition to described experimenter, and wherein, described siRNA composition reduces the expression of the nucleotide sequence that is selected from RCC 1-251.

24. method for the treatment of or preventing renal cell carcinoma among the experimenter, comprise antibody or the segmental step of its immunologic competence of described experimenter being used pharmacy effective dose, described antibody or its immunologic competence fragment with by the protein binding of the arbitrary genes encoding that is selected from RCC 1-251.

25. the method for renal cell carcinoma among treatment or the prevention experimenter, it comprises uses vaccine to described experimenter, and described vaccine comprises: (a) by the polypeptide of the nucleic acid encoding that is selected from RCC 1-251; (b) the immunologic competence fragment of described polypeptide; Or (c) polynucleotide of coding said polypeptide.

26. the method for the treatment of or preventing renal cell carcinoma among the experimenter, it comprises that to described experimenter's administered compound described compound strengthens the expression of the polynucleotide that are selected from RCC 252-972, perhaps strengthens the activity by its encoded polypeptides.

27. the method for the treatment of or preventing renal cell carcinoma among the experimenter, described method comprise the step of using the compound that obtains according to each method among the claim 15-19.

28. the method for renal cell carcinoma among treatment or the prevention experimenter, it comprises the medicine of described experimenter being used pharmacy effective dose, and described medicine contains (a) and is selected from the polynucleotide of RCC 252-972 or (b) by the polypeptide of this polynucleotide encoding.

29. the composition for the treatment of or preventing renal cell carcinoma, described composition contains the antisense polynucleotides or the siRNA of pharmacy effective dose, and described antisense polynucleotides or siRNA are at the polynucleotide that are selected from RCC 1-251.

30. the composition of treatment or prevention renal cell carcinoma, described composition contains pharmacy effective dose antibody or its fragment, described antibody or its fragment with by the protein binding of the genes encoding that is selected from RCC 1-251.

31. the composition for the treatment of or preventing renal cell carcinoma, described composition contain the compound that passes through each method selection among the claim 15-19 of pharmacy effective dose as activeconstituents, and pharmaceutically acceptable carrier.

32. the method for the treatment of or preventing the renal cell carcinoma among the experimenter comprises to described experimenter and uses the composition that comprises siRNA (siRNA) that described siRNA suppresses the expression of C6700V2, B7032N or B9320 V2.

33. the method for claim 32, wherein, described siRNA include the phosphorothioate odn sequence and with anti sense nucleotide sequence from the hybridization of the sequence-specific of C6700V2, B7032N or B9320V2.

34. the method for claim 32, wherein, described kidney is renal cell carcinoma (RCC).

35. the method for claim 33, wherein, described siRNA comprises corresponding to the ribonucleoside acid sequence that is selected from SEQ IDNO:43,47,81,106 or 110 sequence as target sequence.

36. the method for claim 35, wherein said siRNA has general formula 5 '-[A]-[B]-[A ']-3 ', in the formula

[A] is the ribonucleoside acid sequence, and it is corresponding to the sequence that is selected from Nucleotide shown in the SEQ ID NO:43,47,81,106 or 110,

[B] be the ribonucleotide ring sequence formed by 3-23 Nucleotide and

The ribonucleoside acid sequence that the complementary sequence of [A '] serve as reasons [A] is formed.

37. the method for claim 32, described composition comprises the transfection toughener.

38. comprise the duplex molecule of sense strand and antisense strand, wherein, described sense strand contains corresponding to the ribonucleoside acid sequence that is selected from SEQ ID NO:43,47,81,106 or 110 target sequence, and wherein said antisense strand contains and described sense strand complementary ribonucleoside acid sequence, and wherein said sense strand and described antisense strand mutual cross mutually form described duplex molecule; And when wherein described duplex molecule being imported the cell of expressing C6700V2, B7032N or B9320 V2 gene, described duplex molecule suppresses described expression of gene.

39. the duplex molecule of claim 38, wherein, described target sequence contain from be selected from SEQ IDNO:65,112 or 118 nucleotide sequence at least about 10 successive Nucleotide.

40. the duplex molecule of claim 39, wherein, described target sequence contains from about 19～about 25 the successive Nucleotide that are selected from SEQ IDNO:65,112 or 118 nucleotide sequence.

41. the duplex molecule of claim 40, wherein said duplex molecule are to comprise by the sense strand of strand ribonucleoside acid sequence binding and the single ribonucleotide transcript of antisense strand.

42. the duplex molecule of claim 39, wherein said duplex molecule are the oligonucleotide that length is less than about 100 Nucleotide.

43. the duplex molecule of claim 42, wherein said duplex molecule are the oligonucleotide that length is less than about 75 Nucleotide.

44. the duplex molecule of claim 43, wherein said duplex molecule are the oligonucleotide that length is less than about 50 Nucleotide.

45. the duplex molecule of claim 44, wherein said duplex molecule are the oligonucleotide that length is less than about 25 Nucleotide.

46. the oligonucleotide of the duplex molecule of claim 45, wherein said duplex molecule about 19～about 25 Nucleotide that are length.

47. a carrier, the duplex molecule of its coding claim 39.

48. the carrier of claim 47, wherein said vector encoded has the transcript of secondary structure, and comprises sense strand and antisense strand.

49. the carrier of claim 48, wherein, described transcript further comprises the strand ribonucleoside acid sequence that connects described sense strand and described antisense strand.

50. carrier, it contains the polynucleotide of the combination that comprises sense strand nucleic acid and antisense strand nucleic acid, wherein, described sense strand nucleic acid comprises and is selected from SEQ ID NO:43,47,81,106 or 110 nucleotide sequence, and described antisense strand nucleic acid is by forming with described sense strand complementary sequence.

51. the carrier of claim 49, wherein, described polynucleotide have general formula 5 '-[A]-[B]-[A ']-3 ', in the formula

[A] is SEQ ID NO:43,47,81,106 or 110 nucleotide sequence;

[B] is the nucleotide sequence of being made up of 3-23 Nucleotide,

[A '] be and [A] complementary nucleotide sequence.

52. the pharmaceutical composition of treatment or prevention renal cell carcinoma, its siRNA (siRNA) of expression that contains inhibition C6700V2, the B7032N of pharmacy effective dose or B9320 V2 is as activeconstituents, and pharmaceutically acceptable carrier.

53. the pharmaceutical composition of claim 51, wherein, described siRNA comprises and is selected from SEQ ID NO:43,47,81,106 or 110 nucleotide sequence as target sequence.

54. the composition of claim 52, wherein, described siRNA has general formula 5 '-[A]-[B]-[A ']-3 ', in the formula

[A] is the ribonucleoside acid sequence corresponding to nucleotide sequence shown in the SEQ ID NO:43,47,81,106 or 110,

[B] be the ribonucleoside acid sequence formed by 3-23 Nucleotide and

[A '] is the complementary ribonucleoside acid sequence with [A].

Images