CN1299829A - New human cell cycle regulating protein and its code sequence and use - Google Patents

Abstract

The present invention relates to a novel cell division control protein 2, called D-cdc 2 for short. Said invention provides polynucleotide for coding said protein molecule and method for producing said protein molecule by means of recombination technology. Said invention also discloses the application of polynucleotide for coding this novel cell division control protein molecule. Said invention also discloses the tactics of resisting said protein molecule of diagnosing and curing disease, in particular for diagnosing and curing tumor, nervous system and angiocardiopathy.

Description

New D-CDC2, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding D-CDC2 D-cdc2, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new intracellular protein relevant with neurocyte proliferation with cell proliferation, particularly tumour cell.

Keep the basis that normal cell cycle kinetics is all biological phenomenas, the relation of their regulation and control and the generation of regulation and control obstacle and disease is unusual active research field in the life science in recent years.Each biochemical event of cell cycle be subjected to the proteic strict regulation and control of series, various cyclin white matters are expressed appearance during in chronological order with specific cells mutually.

The breakthrough of the study on regulation of cell cycle in recent years be numerous cyclin (cyclin), cyclin dependent kinase (cyclin dependent kinases, CDK) and the discovery of phytokinin cell cycle regulation protein moleculars such as (septin) and corresponding gene.Cell cycle regulating is extremely complicated, relate to of the accumulation of different cycles element at different checkpoints of cell cycle (check-point), with different cell cycle gene (cell devision cycle, cdc) interactions of gene expression product and related protein kinase thereof.Cyclin-CDKs and septins equimolecular in the coordination cycle such as dna replication dna and separate, the duplicating and separate critical events such as the formation of spindle body of centrosome.(Nurse?P,et?al.Nat?Med?1998；4(10):1103-6)

People use may acting on of cdc gene that various temperature sensitive strain yeast saccharomyces cerevisiae (saccharomyces cereislac) determined at first that in cell division cycle some and cell cycle advance relevant and order of representation and these genes.Kind surplus present known yeast cdc gene has 70, many genes are cloned and are checked order, it is now know that, and duplicating by the cdc31 gene of spindle body controlled, that begin that major control point works in the cell cycle is cdc28, and the DNA that cdc8 acted in chromosome cycle is synthetic, and cdc24 controls sprouting of cytoplasmic cycle.Required protein or enzyme in some genes encoding cell cycle secondary process in addition, as dna ligase (cdc9), dna polymerase i (cdc17) etc., these genes are undergone mutation and can be made cell the phenotype of cell cycle occur stopping.

Phytokinin (septin) (Cooper JA, et al.J Cell Biol 1996; 134 (6): 1345-8; Field CM, Kellogg D, Trends Cell Biol, 1999; 9 (10): 387-94) belong to and have the active conservative protein of GTPase family.This family protein comprises the cell divising regulatory protein of a plurality of GTP of having enzymic activitys: H5, NEDD5, CDC10, hCDCrel and AF17q25 etc.They participate in many cell functions, move the change of (cytokinesis) and cell shape as specialization, the cytoplasm in cell surface zone, participate in to form novel shell system or for the assembling of signal transduction mixture provides support, thereby participate in cytokinesis and zymic sprout (budding).Constituted around newly the sprout silk ribbon of 10nm thickness of neck of yeast as them.These albumen can with the lamin (nuclear lamin) (phosphorylation may be relevant with the fragmentation of nuclear membrane) that constitutes stratum nucleare plate (nuclear lamina), the chromosomin (relevant) that comprises histone, tubulin (forming relevant), Actin muscle etc. with spindle body with the karyomit(e) cohesion.

A kind of phosphorprotein with transcriptional regulatory activity of cdc10 genes encoding, its regulation and control target gene is cdc18.In fission yeast, during the cdc10 transgenation, the cell growth just is stopped in before the starting point.When cell passed through starting point, cdc10 started the cdc18 genetic transcription, and its transcription product orders about cell and enters the S phase, and suppressed the initial of M phase; The cell of disappearance cdc18 can not synthetic DNA, but cell enters abnormal mitotic division.Start one section sequence homology in two kinds of transcriptional regulator Swi4 joining type and the cdc10 gene in the blastogenesis yeast; Blastogenesis yeast CDC6 then with the cdc18 gene in another section sequence homology, the CDC6 gene is initial relevant with the S phase.AF17q25 and hCDCrel may participate in 11q23 relevant leukemic generation (Taki T, et al.CancerRes 1999; 59:4261).Nedd5, CDCrel-1 may participate in the secretion of neurotransmitter and the regulation and control that efflux (Beites CL, et al.Nat Neurosci 1999; 2:434).

The disorder of cell cycle can cause the no control growing propagation of cell.Form Study on Mechanism for cell hyperproliferation disease (as tumour, atherosclerosis etc.) at present, just more and more closely the regulation and control with the cell cycle combine.The influence of cell cycle can just produce/the negative regulation effect by regulation and control cdc gene or proteic expression.Just regulation and control factor of cell cycle comprises oncoprotein, hormone, cell growth factor and cell cycle internal composition, wherein the most important thing is maturation promoting factor (MPF), and it is dimer (cyclin and a kinases), can make the substrate phosphorylation dephosphorylation.Relate to cancer suppressor protein and CDKs inhibitor etc. in the negative regulation factor.By interfering the cell cycle kinetics process, may provide new molecular therapy pattern for the cell hyperproliferation prevention and treatment of diseases.Enter the S phase as cell cycle regulating factor p21 and p27 are capable of inhibiting cell by the G1 phase, thereby suppress the propagation of vascular endothelial cell and tumour cell, in antitumor and anti-cardiovascular disease, have the I clinical value.

Research shows that unusual the or disappearance of cell cycle regulating protein is relevant with numerous disease, and therefore, to research and develop D-CDC2 significant for therapeutic purpose.

The purpose of this invention is to provide a kind of new D-CDC2-D-cdc2 albumen with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.D-cdc2 albumen of the present invention is a kind of septin2 homolgous molecule.

In a first aspect of the present invention, novel isolated D-cdc2 polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people D-cdc2 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 127-1680 position among the SEQ ID NO:1; (b) has the sequence of 1-3018 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people D-cdc2 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human D-cdc2, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people D-cdc2 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people D-cdc2 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-3018 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people D-cdc2 polypeptide active is provided, and the compound that suppresses people D-cdc2 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people D-cdc2 polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of D-cdc2 in the test sample, it comprises: sample is contacted with the proteic specific antibody of D-cdc2, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample D-cdc2 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people D-cdc2 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people D-cdc2 polypeptide active, and perhaps screening suppresses the antagonist of people D-cdc2 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people D-cdc2 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people D-cdc2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour, neural system and cardiovascular diseases.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the cDNA sequence of people D-cdc2 of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization.

Fig. 2 is the proteic full length amino acid sequence of people D-cdc2 of the present invention.

Fig. 3 is people D-cdc2 albumen of the present invention and the proteic amino acid sequence homology comparison diagram of setpin2 (accession number is P39827).The top sequence is people D-cdc2, and the below sequence is a septin2 albumen.Identical amino acid marks with " | " between two sequences, and similar amino acid marks with ": " or ". ".

Fig. 4 is the hydrophobicity graphic representation of the proteic Kyte-doolitte hydrophobicity analysis of people D-cdc2 of the present invention.

In the present invention, term " D-cdc2 albumen ", " D-cdc2 polypeptide " or " cell cycle regulating protein D-cdc2 " be used interchangeably, all refer to have human cell cycle regulating protein D-cdc2 amino acid sequence (SEQ ID NO:2) albumen or polypeptide. They comprise the cell cycle regulating protein D-that contains or do not contain initial methionine Cdc2.

As used herein, " separation " refers to that material separates (if natural thing from its primal environment Matter, primal environment namely are natural surroundingses). Such as the polynucleotide under the native state in the active somatic cell and polypeptide be Do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist Separately, then for separation and purification.

As used herein, it is natural that " D-cdc2 albumen or the polypeptide of separation " refers to that the D-cdc2 polypeptide is substantially free of Relative other albumen, lipid, carbohydrate or other material. Those skilled in the art can use the albumen of standard Matter purification technique purifying D-cdc2 albumen. Basically pure polypeptide can produce on non-reduced polyacrylamide gel Single master tape. The purity of D-cdc2 polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide. This Bright polypeptide can be the product of natural purifying, or the product of chemical synthesis, or uses recombinant technique from protokaryon Or produce in the eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell). According to The host that the recombinant production scheme is used, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated. Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analog of people D-cdc2 albumen. As used herein, term " sheet Section ", " derivative " refer to basically keep natural human D-cdc2 albumen of the present invention identical with " analog " Biological function or active polypeptide. Polypeptide fragment of the present invention, derivative or analog can be that (ⅰ) has one Or a plurality of conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and like this The amino acid residue of replacement can be also can be by the genetic code coding, or (ⅱ) at one or more ammonia Base has the polypeptide of substituted radical in the sour residue, or (ⅲ) mature polypeptide and another compound (such as prolonging polypeptide partly The compound of phase, for example polyethylene glycol decline) merge formed polypeptide, or (ⅳ) additional amino acid sequence is fused to This peptide sequence and the polypeptide that forms are (such as targeting sequencing or secretion sequence or be used for sequence or the albumen of this polypeptide of purifying Former sequence, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, spread out Biological and analog belongs to the known scope of those skilled in the art.

In the present invention, term " people D-cdc2 polypeptide " refers to have the SEQ ID NO. of people D-cdc2 protein active The polypeptide of 2 sequences. This term also comprise have with people D-cdc2 albumen identical function, SEQ ID NO.2 order The variant form of row. These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and (being generally in 20, preferably is in 10, better for one of the terminal interpolation of C end and/or N or several Ground is in 5) amino acid. For example, in the art, the amino acid close or similar with performance replaces The time, the common function that can not change protein. Again such as, add one or several in that C end and/or N are terminal Amino acid also can not change the function of protein usually. This term also comprise people D-cdc2 albumen active fragment and Reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural sudden change Body, induced mutation body, under high or low stringency condition, can compile with the DNA of people D-cdc2 DNA hybridization The albumen of code and the polypeptide or the albumen that utilize the antiserum acquisition of anti-people D-cdc2 polypeptide. The present invention also provides Other polypeptide, as comprise the fusion of people D-cdc2 polypeptide or its fragment. Except the polypeptide of total length almost, The present invention has also comprised the soluble fragments of people D-cdc2 polypeptide. Usually, this fragment has people D-cdc2 polypeptide order Row at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 Continuous amino acid is more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analog of people D-cdc2 albumen or polypeptide. These analogs and natural human D-cdc2 polypeptide Difference can be difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, or The person haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can be by various skills Art obtains, as by radiation or be exposed to mutagens and produce random mutagenesis, and also can be by direct mutagenesis method or other The biological technology of known molecular. Analog also comprises having and is different from the amino acid whose residue of natural L-(such as D-amino Acid) analog, and the analog with that non-natural exists or synthetic amino acid (such as β, gamma-amino acid). Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(usually the not changing primary structure) form of modification comprises: chemically derived form such as the second of the polypeptide that body is interior or external Acidylate or carboxylated. Modify and also to comprise glycosylation, such as those in the synthetic and processing of polypeptide or further add work step Carry out glycosylation modified in rapid and polypeptide that produce. This modification can by polypeptide is exposed to carry out glycosylated Enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishing. Modified forms also comprises having phosphorylation amino The sequence of acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprising being modified improves Its anti-proteolysis performance or optimized the polypeptide of solubility property.

In the present invention, " people D-cdc2 albumen conservative variation polypeptide " refers to the amino acid with SEQ ID NO:2 Sequence is compared, and has 10 at the most, and preferably at the most 8, more preferably at the most 5,3 amino acid at the most best Replaced by similar performance or close amino acid and form polypeptide. These conservative variation polypeptide are preferably according to table 1 Carry out amino acid substitution and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Ash	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Ash
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Mla；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Polynucleotides of the present invention can be dna form or rna form. Dna form comprises cDNA, base Because of group DNA or artificial synthetic DNA. DNA can be strand or double-stranded. DNA compiles Code chain or noncoding strand. The coding region sequence of encoding mature polypeptide can with the code area shown in the SEQ ID NO:1 The variant of the identical or degeneracy of sequence. As used herein, " variant of degeneracy " refers to compile in the present invention Code has the protein of SEQ ID NO:2, but with the differentiated nuclear of coding region sequence shown in the SEQ ID NO:1 Acid sequence.

The polynucleotides of the mature polypeptide of coding SEQ ID NO:2 comprise: the coded sequence of an encoding mature polypeptide: The coded sequence of mature polypeptide and various additional code sequence; The coded sequence of mature polypeptide is (with optional additional volume The code sequence) and non-coding sequence.

Term " polynucleotides of coded polypeptide " can be the polynucleotides that comprise this polypeptide of encoding, and also can be The polynucleotides that also comprise additional code and/or non-coding sequence.

The invention still further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention Polypeptide or fragment, analog and the derivative of polypeptide. The variant of these polynucleotides can be natural generation etc. The variant that position variant or non-natural take place. These nucleotide diversity bodies comprise replacement variant, deletion mutation body With the insertion variant. As known in the art, allelic variant is the replacement form of polynucleotides, and it may Replacement, disappearance or the insertion of one or more nucleotides, but can be from the merit of the polypeptide that changes in fact its coding Energy.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.: or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation encoding D-cdc2.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

People D-cdc2 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or D-cdc2 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the D-cdc2 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people D-cdc2 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, people D-cdc2 polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people D-cdc2 DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Go into phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people D-cdc2 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism D-cdc2 protein function as pharmacological agent D-cdc2 protein function.The peptide molecule that can suppress or stimulate people D-cdc2 protein function that can be used for seeking therapeutic value with the recombinant human D-cdc2 protein screening peptide library of expressing.

On the other hand, the present invention also comprises people D-cdc2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people D-cdc2 gene product or fragment.Preferably, refer to that those can combine with people D-cdc2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people D-cdc2, comprise that also those do not influence the antibody of people D-cdc2 protein function.The present invention also comprise those can with modify or without the people D-cdc2 gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people D-cdc2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human D-cdc2 albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people D-cdc2 protein function and the antibody that does not influence people D-cdc2 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people D-cdc2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people D-cdc2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people D-cdc2 can be used in the immunohistochemistry technology, detects the people D-cdc2 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people D-cdc2, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people D-cdc2 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people D-cdc2 or activity.

Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people D-cdc2 albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell (for example lymph-node cell etc.) of people D-cdc2 protein positive.

The production of polyclonal antibody can choose D-cdc2 albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with D-cdc2 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of tumour, neural system and cardiovascular diseases aspect.When using D-cdc2 albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.

The present invention also provides a kind of pharmaceutical composition, and it contains D-cdc2 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the D-cdc2 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of people D-cdc2 also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of D-cdc2 of the proteic nothing expression of D-cdc2 or unusual/non-activity.The D-cdc2 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic D-cdc2 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the D-cdc2 transgenosis to cell.The method that structure carries the recombinant viral vector of D-cdc2 gene is found in existing document (Sambrook, et al.).Recombinant human D-cdc2 gene can be packaged in the liposome in addition, and then is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people D-cdc2 mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people D-cdc2 obtains.During screening, must carry out mark to people D-cdc2 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people D-cdc2 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people D-cdc2 protein level that is detected in the test can be with laying down a definition the importance of people D-cdc2 albumen in various diseases and be used to the disease of diagnosing D-cdc2 albumen to work.

Whether having the proteic method of D-cdc2 in a kind of detection test sample is to utilize the proteic specific antibody of D-cdc2 to detect, and it comprises: sample is contacted with the D-cdc2 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample D-cdc2 albumen.

The proteic polynucleotide of D-cdc2 can be used for the diagnosis and the treatment of D-cdc2 protein related diseases.Aspect diagnosis, the proteic polynucleotide of D-cdc2 can be used for detecting the proteic expression of D-cdc2 D-cdc2 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of D-cdc2 as the D-cdc2 dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of D-cdc2 albumen and also can detect the proteic transcription product of D-cdc2.

The sudden change that detects the D-cdc2 gene also can be used for the disease of diagnosing D-cdc2 albumen relevant.The form of D-cdc2 protein mutation comprises that the point mutation compared with normal wild type D-cdc2 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of D-cdc2 prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendellan Inheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 3018 bases, and its open reading frame is positioned at the 127-1680 position, and the coding total length is 517 amino acid whose people D-cdc2 albumen (SEQ IDNO:2).This D-cdc2 albumen belongs to the cell cycle regulating protein family molecule, and with cdc10, septin2 aminoacid sequence height homology, consistence can be up to 70%, and similarity then can reach 83%.In addition, D-cdc2 albumen also comprises GTP binding motif (GX4GKS-DX2G-KXD).The Northern engram analysis shows wide expression in tissue.

The research carried out prompting, D-cdc2 may be the target protein of interfering the cell cycle, can technology such as build by a minute submodule, competitive its short cell fission effect of blocking-up, the effect of the antitumor and anti-cardiovascular disease of tool potential.Therefore, D-cdc2 albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, neural system and cardiovascular diseases new treatment approach are provided, thereby have great application prospect.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the clone of people D-cdc2 cDNA

Extract the total RNA of human dendritic cell with Trizol (Gibco company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is inserted into cDNA fragment orientation on the multiple clone site of carrier with SuperScript II clone's test kit (available from Gibco), transforms DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID No:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as D-CDC2 D-cdc2, its encoding gene called after D-CDC2 D-cdc2 gene.

Sequence SEQ ID NO:1 total length is 3018bp, comprises 3 ' end non-coding region of 5 of 126bp ' end non-coding region and 1338bp, and coding contains 517 amino acid whose polypeptide.Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 60kD.

They are different with known for the BLAST analysis revealed, and with people septin2 aminoacid sequence height homology, consistence reaches 70%, and similarity reaches 83% (Fig. 3), belongs to the cell cycle regulating protein family molecule.In addition, also comprise a GTP binding motif (GX4GKS-DX2G-KXD) in D-cdc2 albumen, this also further points out it may participate in the regulation and control of cell cycle.Hydrophobicity analysis shows that D-cdc2 is an intracellular protein (Fig. 4).Embodiment 2: with the encoding sequence of RT-PCR method human cloning D-cdc2

Be in logarithmic phase B lymphoma cell strain Raji cell total rna with Trizol (Gibco company) extraction, get 6mg cell total rna and 0.5 μ g Oligo-dT ₁₂- ₁₈Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ l ddH after finishing ₂O dilutes.The used primer of pcr amplification is as follows: have adopted primer 5 '-cggaggcagcctagcctcgcgccccgcccg-3 ' (SEQ ID NO:3), antisense primer 5 '-gtgctatgcaaggttttatttacaactt-3 ' (SEQ ID NO:4).The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 1-3001 position shown in the SEQ ID NO:1 are identical.The Northern engram analysis of embodiment 3 D-cdc2

Carry out the Northern trace by following ordinary method: filter membrane to be checked places the hybridization solution of 10ml through 68 ℃ of preheatings, in hybrid heater (Bellco) in 68 ℃ of prehybridizations 30 minutes; The cDNA probe that mark is good was in 95～100 ℃ of sex change 2～5 minutes, and (cDNA probe final concentration is 2～10ng/ml or 1～2 * 10 to put rapid cooling back adding hybridization solution on ice ⁶Cpm/ml), fully mixing was hybridized 2 hours in 68 ℃.After hybridization finishes, filter membrane with 2 * SSC, the drip washing of 0.05%SDS room temperature for several times, the washing lotion several is changed in continuing vibration flushing 30～40 minutes therebetween.Use 0.1 * SSC, 0.1%SDS in 50 ℃ of vibration flushings 20～40 minutes subsequently.Last filter membrane wrapped up with plastic fresh-keeping membrane, in-70 ℃ of exposure X line films 24～48 hours.

Northern blot hybridization result shows: in many healthy tissuess such as liver, spleen, peripheral blood, brain expression is arranged all, this shows that D-cdc2 albumen is a kind of albumen of wide expression.Embodiment 4 people D-cdc2 are recombinant expressed

In this embodiment, be template with the pcr amplification product among the embodiment 2, increase with the PCR Oligonucleolide primers of 5 ' and 3 following ' end of sequence, obtain people D-cdc2 DNA as inserting fragment.

5 ' end Oligonucleolide primers sequence of using in the PCR reaction is:

5′-cgggatccATGGCCTCCTCCGAGGTGG-3′(SEQ?ID?NO:5)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is the part encoding sequence that is begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5′-ggaattctcattcactctggcttatg-3′(SEQ?ID?NO:6)

This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people D-cdc2 of EcoR I restriction enzyme.

The PCR product purification that obtains after cutting, BamH I/EcoR I enzyme is organized and is converted into the competence bacillus coli DH 5 alpha more according to a conventional method with plasmid pUC18, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The people D-cdc2 cDNA BamH I/EcoR I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia company), forms carrier pGEX-2T-D-cdc2, be transformed into bacillus coli DH 5 alpha then.

Choosing the positive DH5 α clone who expresses D-cdc2 is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7 mM KCl, 10.1mM Na ₂HPO ₄, 1.8mMKH ₂PO ₄PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10 mM gsh, 50 mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, and obtain N and hold the people D-cdc2 fusion rotein that contains GST, obtain D-cdc2 albumen after thrombin (Sigma) enzyme cuts except that GST, molecular weight is about 60kD.

Carry out the n terminal amino acid sequential analysis with the Edams hydrolysis method, confirmed that its N terminal sequence conforms to the N terminal sequence shown in the SEQ ID NO:2.Embodiment 5: anti-people D-cdc2 production of antibodies

The recombinant protein people D-cdc2 that obtains among the embodiment 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people D-cdc2 gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information:

(ⅱ) denomination of invention: new D-CDC2, its encoding sequence and purposes

(ⅲ) sequence number: the information of 6 (2) SEQ ID NO:1: (ⅰ) sequence signature:

(A) length: 3018bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO:1：CGGAGGCAGC CTAGCCTCGC GCCCCGCCCG TTGCTTCTGC CCTCCGGCCT TCCCGCCGCC 60GTCGCCGGGA CCAGCCGCTC GGGGCCGGGC TGATACAGCC GCTTCACCGT GCCCCTGCCC 120GCGACCATGG CCTCCTCCGA GGTGGCGCGG CACCTGCTCT TTCAGTCTCA CATGGCAACG 180AAAACAACTT GTATGTCTTC ACAAGGATCA GATGATGAAC AGATAAAAAG AGAAAACATT 240CGTTCGTTGA CTATGTCTGG CCATGTTGGT TTTGAGAGTT TGCCTGATCA GCTGGTGAAC 300AGATCCATTC AGCAAGGTTT CTGCTTTAAT ATTCTCTGTG TGGGGGAAAC TGGAATTGGA 360AAATCAACAC TGATTGACAC ATTGTTTAAT ACTAATTTTG AAGACTATGA ATCCTCACAT 420TTTTGCCCAA ATGTTAAACT TAAAGCTCAG ACATATGAAC TCCAGGAAAG TAATGTTCAA 480TTGAAATTGA CCATTGTGAA TACAGTGGGA TTTGGTGACC AAATAAATAA AGAAGAGAGC 540TACCAACCAA TAGTTGACTA CATAGATGCT CAGTTTGAGG CCTATCTCCA AGAAGAACTG 600AAGATTAAGC GTTCTCTCTT TACCTACCAT GATTCTCGCA TCCATGTGTG TCTCTACTTC 660ATTTCACCGA CAGGCCACTC TCTGAAGACA CTTGATCTCT TAACCATGAA GAACCTTGAC 720AGCAAGGTAA ACATTATACC AGTGATTGCC AAAGCAGATA CGGTTTCTAA AACTGAATTA 780CAGAAGTTTA AGATCAAGCT CATGAGTGAA TTGGTCAGCA ATGGCGTCCA GATATACCAG 840TTCCCAACGG ATGATGACAC TATTGCTAAG GTCAACGCTG CAATGAATGG ACAGTTGCCG 900TTTGCTGTTG TGGGAAGTAT GGATGAGGTA AAAGTCGGAA ACAAGATGGT CAAAGCTCGC 960CAGTACCCTT GGGGTGTTGT ACAAGTGGAA AATGAAAACC ACTGTGACTT TGTAAAGCTG 1020CGGGAAATGC TCATTTGTAC AAATATGGAG GACCTGCGAG AGCAGACCCA TACCAGGCAC 1080TATGAGCTTT ACAGGCGCTG CAAACTGGAG GAAATGGGCT TTACAGATGT GGGCCCAGAA 1140AACAAGCCAG TCAGTGTTCA AGAGACCTAT GAAGCCAAAA GACATGAGTT CCATGGTGAA 1200CGTCAGAGGA AGGAAGAAGA AATGAAACAG ATGTTTGTGC AGCGAGTAAA GGAGAAAGAA 1260GCCATATTGA AAGAAGCTGA GAGAGAGCTA CAGGCCAAAT TTGAGCACCT TAAGAGACTT 1320CACCAAGAAG AGAGAATGAA GCTTGAAGAA AAGAGAAGAC TTTTGGAAGA AGAAATAATT 1380GCTTTCTCTA AAAAGAAAGC TACCTCCGAG ATATTTCACA GCCAGTCCTT TCTGGCAACA 1440GGCAGCAACC TGAGGAAGAG GACAAGGACC GTAAGAACTC CAATTTTTTG TAAAACAGAA 1500GTTCCAGAGC ACAGAAGGTC ATCATCACAA GCAAACTTTA TTAAAAAAAA ACTAGAAGTG 1560TGCTTTGATT TTGCTGTTAT TTGTTTTATC ACTTCTATAT TTGGTGAACA GCCACAGTTA 1620CTGATATTTA TGGAAAAGTA CTTTCAAGTA CAAGGTCAAT ACATAAGCCA GAGTGAATGA 1680TACTACAAGT TGAGCATCTC TAATTCAAAA ATCTGAAATC CAGAAGCTTC AAAATCTGAA 1740TCTTTTTGAG CACTGACTTG ACCCCACAAG TGGAAAATTC CCCACCCGAC ACCTTTGCTT 1800TCTGATGGTT CAGTTTAAAC AGATTTTGTT TCTTGCACAA AATTTTTGTA TAAATTACTT 1860TCAGGCTATA TGTATAAGGT GGATGTGAAA CATGAATTAT GTAATTAGAG TCGGGTCCCG 1920TTGTGTATAT GCAGATATTC CAAACCTGAA ATCCAAAACA CTTCTGGTCC CTAGCATTTT 1980GGATAAGGGA TACTCAGCTT GTACCTATAT ATTCATATAT ATTCACTGTT GTTAGAAATG 2040TTTAAGTTGC TGTTCTGTGA TGAATCTAAA TCTTTTCTCT TGCTACCAAG CTATTGTCAC 2100TGCAGTGCAT TATACCAAAG AGCGAAGTCA GTGCCACTGA AAATACAGAA CCCATTAATA 2160TCGTGGCTAT CTGATTACAT TTATATTCCA AGATGAACCT TTTTTATATA TGCTAAAAAT 2220TTTGGGGAAT ATGTTTTGGG ATGTATTATG GAGCTAAAAC TCTAACCTCT TAATAGTTTT 2280ATAGAACTTA AAAATTTTTT ATACAATTAC CCAATTGGTG ATATGATCTT AAGCTTTTGT 2340GTCAGATTAT TTAATATGAT GACTTCATGC TTTATTATGC CTTATTATGG CTGACGTATT 2400ACTGTGGTGA AACAAAATAT CTTTAAAAGT TAAAACATCC AGATATATAA GCTATTTTTT 2460CCTAAGGATA AAGTACCTTT GAGCATGAGT GTATCACAGC TTTCATTAGG AAAACTTTTC 2520ATTACATACT TGTTTAAACT CTGTCTTCCA GGGTAAAAAT AATAAGGTTG AATCATTTTA 2580TTAAAAATAC TTTTTAAGAA AATAACTATG AACATCTGAA TATTAAAGAT ATAAAAATGC 2640ACATAATTCA TATTTCAGGT GGTATTTGCA TTCAGTGCCT TACTGGTATT CTCAGAACAT 2700TTTAATGATT TCTAACATTT CTTAACAGTC ATAGATATAT ACATTTTCAT TTTTTGTACT 2760TGAATATTCT AAATAAAACT GACATTTACT CTTGACAAAT AAAACATATA TTTACTAAAA 2820TGTGTTTAAT TTTCCTTTCT GAAAACTCTC ATTTTAAAAA CGTTCATTTA ATTATGTATT 2880TGAATTATTT TGGAGATGAG GTATTTTATG AGTATTTTCA GACAATGAAA CTTATTAGTC 2940TGTGTCAGAT TCTGAGCAAT CATAGAGTCA TCTAAGTTGT AAATAAAACC TTGCATAGCA 3000CAAAAAAAAA AAAAAAAA 3018 ( 2 ) SEQ ID NO:2：

(ⅰ) sequence signature:

(A) length: 517 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

( ⅹⅰ ) ：SEQ ID NO:2:MASSEVARHL LFQSHMATKT TCMSSQGSDD EQIKRENIRS LTMSGHVGFE 50SLPDQLVNRS IQQGFCFNIL CVGETGIGKS TLIDTLFNTN FEDYESSHFC 100PNVKLKAQTY ELQESNVQLK LTIVNTVGFG DQINKEESYQ PIVDYIDAQF 150EAYLQEELKI KRSLFTYHDS RIHVCLYFIS PTGHSLKTLD LLTMKNLDSK 200VNIIPVIAKA DTVSKTELQK FKIKLMSELV SNGVQIYQFP TDDDTIAKVN 250AAMNGQLPFA VVGSMDEVKV GNKMVKARQY PWGVVQVENE NHCDFVKLRE 300MLICTNMEDL REQTHTRHYE LYRRCKLEEM GFTDVGPENK PVSVQETYEA 350KRHEFHGERQ RKEEEMKQMF VQRVKEKEAI LKEAERELQA KFEHLKRLHQ 400EERMKLEEKR RLLEEEIIAF SKKKATSEIF HSQSFLATGS NLRKRTRTVR 450TPIFCKTEVP EHRRSSSQAN FIKKKLEVCF DFAVICFITS IFGEQPQLLI 500FMEKYFQVQG QYISQSE 517 ( 2 ) SEQ ID NO:3

(ⅰ) sequence signature

(A) length: 30 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3:CGGAGGCAGC CTAGCCTCGC GCCCCGCCCG 30 (2) SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4GTGCTATGCA AGGTTTTATT TACAACTT 28 (2) SEQ ID NO:5

(ⅰ) sequence signature

(A) length: 27 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5:CGGGATCCAT GGCCTCCTGC GAGGTGG 27 (2) SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 26 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:6:GGAATTCTCA TTCACTCTGG CTTATG 26

Claims (14)

1. an isolating people D-cdc2 polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 127-1680 position among the SEQ ID NO:1;

(b) has the sequence of 1-3018 position among the SEQ ID NO:1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. preparation method with polypeptide of people D-cdc2 protein-active is characterized in that this method comprises:

(a) under the proteic condition of suitable expressing human D-cdc2, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people D-cdc2 protein-active.

9. energy and the described people D-cdc2 of claim 1 protein-specific bonded antibody.

10. nucleic acid molecule, it contains a successive 10-3018 Nucleotide in the described polynucleotide of claim 3.

11. whether there is the proteic method of D-cdc2 in the test sample, it is characterized in that, comprising:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample D-cdc2 albumen.

12. the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes people D-cdc2 protein-active, and perhaps screening suppresses the antagonist of people D-cdc2 protein-active or is used to the peptide finger print identification.

13. the purposes of a nucleic acid molecule as claimed in claim 10 is characterized in that, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or microarray.

14. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.

Images