CN1193041C - New human protein with the function of inhibiting cancer cell growth and its encoding sequence - Google Patents

Abstract

The present invention discloses a novel human protein with the function of inhibiting cancer, polynucleotide for encoding the polypeptide and a method for preparing the polypeptide by a recombinant technology. The present invention also discloses a method of using the polypeptide to treat various diseases, such as cancers. The present invention also discloses an antagonist of the polypeptide and a therapeutic effect thereof. The present invention also discloses the application of the polynucleotide for encoding the human protein with the function of inhibiting cancer.

Description

People's albumen and encoding sequence thereof with anticancer growth function

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to the proteic polynucleotide of people that new coding has cancer suppressing function, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.

Background technology

The research of people's gene group is international focus at present, removes human chromosome DNA large scale sequencing, outside the method for expressed sequence order-checking (EST), also lacks the screening that begins from function and has the high-throughout method of functional gene.

Cancer is one of principal disease of harm humans health.In order to treat effectively and prophylaxis of tumours, people more and more pay close attention to genetic treatment of tumor at present.Therefore, this area presses for people's albumen and the agonist/inhibitor thereof that development research has cancer suppressing function.

Summary of the invention

The purpose of this invention is to provide the new people's protein polypeptide of a class with cancer suppressing function 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.

In a first aspect of the present invention, novel isolated protein polypeptide with cancer suppressing function is provided, and it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26; Or its conservative property variation polypeptide or its active fragments or its reactive derivative.

Preferably, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:26.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned protein polypeptide with cancer suppressing function of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26.More preferably, the sequence of these polynucleotide is selected from down group: coding region sequence or the full length sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27.

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, the preparation method who prepares the polypeptide of the protein-active with cancer suppressing function is provided, this method comprises: (a) have under the proteic condition of cancer suppressing function suitable the expression, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate the polypeptide of protein-active with cancer suppressing function.

In a fifth aspect of the present invention, provide and above-mentioned protein polypeptide specificity bonded antibody with cancer suppressing function.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-800 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the protein polypeptide and the pharmaceutically acceptable carrier with cancer suppressing function of the present invention of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer and cellular abnormality propagation.

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

Embodiment

The present invention adopts large-scale cDNA clone transfection cancer cells, has on the basis of cancer suppressing action in acquisition, proves new gene through order-checking, further obtains full length cDNA clone.DNA transfection evidence, the albumen with cancer suppressing function of the present invention has the effect that suppresses clone's formation to cancer cells (liver cancer cell), and its inhibiting rate is more than 50% or 50%.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating albumen or polypeptide with cancer suppressing function " is meant that the protein polypeptide with cancer suppressing function is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can have the albumen of cancer suppressing function with the purified technology of protein purifying of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.Purity with protein polypeptide of cancer suppressing function can be used amino acid sequence analysis.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, 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 the proteic fragment of the people with cancer suppressing function, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep natural identical biological function or the active polypeptide of people's albumen with cancer suppressing function of the present invention basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.With PP4277 albumen (in this application, its clone's numbering is adopted in proteinic name) (in this application, its clone numbering is adopted in proteinic name) be example, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.With PP4377 albumen (in this application, its clone's numbering is adopted in proteinic name) (in this application, its clone numbering is adopted in proteinic name) be example, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:6 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:5, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:6.Have the albumen of cancer suppressing function for other, can the rest may be inferred.Have the albumen of cancer suppressing function for other, can the rest may be inferred.

The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

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

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

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 95%, be more preferably 97% 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.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid has the proteic polynucleotide of cancer suppressing function to determine and/or to separate to encode.

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

Dna sequence dna of the present invention can obtain with several method.For example, with hybridization technique DNA isolation well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homology nucleotide sequence and 2) antibody screening of expression library to be to detect the dna fragmentation of the clone with common structure feature.

The proteic specific DNA fragment sequence that coding has cancer suppressing function produces also and can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of required polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.When if required amino acid whose whole sequence is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) mensuration has the level of the proteic transcript of cancer suppressing function; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 15 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, the length of probe within 2kb, preferably is within the 1kb usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of protein gene expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc. with cancer suppressing function.

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 gene of the present invention that obtains as mentioned above, perhaps the available ordinary method of mensuration of the nucleotide sequence of various dna fragmentations etc. such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

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 albumen coded sequence with cancer suppressing function, and the method that produces polypeptide of the present invention through recombinant technology.

By the recombinant DNA technology of routine, can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the protein polypeptide with cancer suppressing function (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). have the proteic polynucleotide of people (or varient) of cancer suppressing function with coding of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

Among the present invention, the people's albumen polynucleotide sequence with cancer suppressing function 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's encoding histone dna sequence dna with cancer suppressing function and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold SpringHarbor 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; Lambda particles phage P _LPromotor; 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 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.Alternative 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.

Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.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's albumen or the polypeptide with cancer suppressing function of reorganization are of use in many ways.These purposes include, but is not limited to: directly have the disease due to the low or forfeiture of the protein function of cancer suppressing function as pharmacological agent and be used to screen and promote or antagonism has antibody, polypeptide or other part of the protein function of cancer suppressing function.For example, antibody can be used for activating or suppressing to have the proteic function of people of cancer suppressing function.The people's protein screening peptide library that has a cancer suppressing function with the reorganization of expressing can be used for seeking the peptide molecule that can suppress or stimulate the people's protein function with cancer suppressing function of therapeutic value.

The present invention also provides screening of medicaments to improve (agonist) or check the method that (antagonist) has the proteic medicament of people of cancer suppressing function to identify.Agonist improves the biological function such as stimulate cellular proliferation of the people's albumen with cancer suppressing function, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the proteic film preparation of people that mammalian cell or expression is had cancer suppressing function is cultivated with the people's albumen with cancer suppressing function of mark.Measure the medicine raising then or check this interactional ability.

The proteic antagonist of people with cancer suppressing function comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The proteic antagonist of people with cancer suppressing function can and be eliminated its function with the people's protein binding with cancer suppressing function, or suppresses to have the proteic generation of people of cancer suppressing function, or combines with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.The proteic antagonist of people with cancer suppressing function can be used for therepic use.

In screening during as the compound of antagonist, the albumen that can have a cancer suppressing function adds during bioanalysis measures, and determines by measuring albumen and the interaction between its acceptor that compounds affect has cancer suppressing function whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.

Polypeptide of the present invention can be directly used in disease treatment, for example, and various malignant tumours and cellular abnormality propagation etc.

Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.

Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound such as TNF etc.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Albumen with cancer suppressing function comes administration with the amount that treats and/or prevents concrete indication effectively.The proteic amount with cancer suppressing function and the dosage range that are applied to the patient will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

The proteic polynucleotide of people with cancer suppressing function also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating since have that the proteic nothing of cancer suppressing function is expressed or the proteic expression with cancer suppressing function of unusual/non-activity due to cell proliferation, growth or metabolic disturbance.The albumen with cancer suppressing function that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic protein-active with cancer suppressing function.For example, a kind of albumen with cancer suppressing function of variation can be the albumen with cancer suppressing function that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the protein expression with cancer suppressing function or the disease of active caused by abnormal.Deriving from the expression vector of virus such as protein gene that retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for having cancer suppressing function is transferred in the cell.The method that structure carries the recombinant viral vector of the protein gene with cancer suppressing function is found in existing document (Sambrook, et al.).The people protein gene of reorganization with cancer suppressing function can be packaged in the liposome and be transferred in the cell in addition.

Suppress to have cancer suppressing function people's protein mRNA oligonucleotide (comprising sense-rna and DNA) 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.

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carry out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension.

The present invention also provides the antibody at the people's proteantigen determinant with cancer suppressing function.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The anti-proteic antibody of people with cancer suppressing function can be used in the immunohistochemistry technology, detects the people's albumen with cancer suppressing function in the biopsy specimen.

With the also available labelled with radioisotope of the protein bound monoclonal antibody of the people with cancer suppressing function, 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 prevents and the relevant disease of people's albumen with cancer suppressing function.The antibody that gives suitable dosage can stimulate or block proteic generation of the people with cancer suppressing function or activity.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As have cancer suppressing function people's albumen high-affinity monoclonal antibody 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 of the people's protein positive with cancer suppressing function.

Available people's albumen or the polypeptide immune animal of the production of polyclonal antibody with cancer suppressing function, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Have cancer suppressing function people's protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the anti-proteic single-chain antibody of people with cancer suppressing function.

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 the people with cancer suppressing function obtains.During screening, must carry out mark to people's protein molecular with cancer suppressing function.

The invention still further relates to quantitatively and detection and localization has the diagnostic testing process of people's protein level of cancer suppressing function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Detected in the test have cancer suppressing function-people's protein level, the disease that can have the importance of people's albumen in various diseases of cancer suppressing function with laying down a definition and be used to diagnose albumen to work with cancer suppressing function.

Proteic polynucleotide with cancer suppressing function can be used for having the diagnosis and the treatment of the protein related diseases of cancer suppressing function.Aspect diagnosis, the proteic polynucleotide with cancer suppressing function can be used for detecting have cancer suppressing function proteic expression whether or under morbid state, have an abnormal exprssion of cancer suppressing function.As the protein D NA sequence with cancer suppressing function can be used for the hybridization of biopsy specimen is had with judgement the proteic abnormal expression of cancer suppressing function.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 the albumen with cancer suppressing function and also can detect proteic transcription product with cancer suppressing function.

The sudden change that detection has the protein gene of cancer suppressing function also can be used for diagnosing the relevant disease of albumen with cancer suppressing function.Form with protein mutation of cancer suppressing function comprises that to have point mutation that the protein D NA sequence of cancer suppressing function compares, transposition, disappearance, reorganization and other any unusual etc. with normal wild type.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).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.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.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manual of BasicTechniques, Pergamon Press, New York (1988).

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, Mendelian 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.

Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.

Pyrenoids thuja acid full length sequence or its fragment with cancer suppressing function of the present invention 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 come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

In addition, because the albumen with cancer suppressing function of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

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 Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The acquisition of embodiment 1:cDNA gene and the restraining effect that the cancer cells clone is formed

PP4277, PP4377, PP4522, PP4583, PP4664, PP4748, PP4762, PP5242, PP5656 obtains by making up the human placenta cDNA library with ordinary method.Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.Make up the cDNA library of above-mentioned mRNA with pCMV-script TMXR cDNA library construction test kit (Stratagene company).Wherein ThermoScript II is used MMLV-RT-Superscript II (GIBCO BRL) instead, and reverse transcription reaction carries out at 42 ℃.Transform XL 10-Gold recipient cell, obtained 1 * 10 ⁶The cDNA library of cfu/ μ g cDNA titre.The first round is picking cDNA clone at random, is probe with high abundance cDNA clone with the cDNA clone who has proved cancer inhibitor cell growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen 96 orifice plate plasmid extraction test kits, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously hepatoma cell line 7721.After the 100ng DNA alcohol precipitation drying, add 6 μ l H ₂Transfection is treated in the O dissolving.Add 0.74 μ l liposome and 9.3 μ l serum-free mediums in every part of DNA sample, behind the mixing, room temperature was placed 10 minutes.Add 150 μ l serum-free mediums in every pipe, divide equally and add 3 holes and grow in 7721 cells of 96 orifice plates, placed 2 hours for 37 ℃, every hole adds 50 μ l serum-free mediums again, 37 ℃ 24 hours.Every hole is changed 100 μ l and is trained liquid entirely, 37 ℃ 24 hours, change the full training liquid 100 μ l that contain G418,37 ℃ 24 ~ 48 hours, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2 ~ 3 times, there is the clone to form up to the microscopy cell, counting.Find that above clone has the cell clone of inhibition formation effect, the result is as shown in the table.

CDNA clone's transfectional cell (7721) clone formation situation

CDNA clones title	C DNA cloning number (three repetitions)			Empty carrier clone number (three repetitions)
							PP4277	0	0	4	45	37	30
PP4377	1	16	4	33	12	16
							PP4522	0	0	0	23	21	14
PP4583	0	0	0	23	21	14
							PP4664	7	6	1	32	34	29
PP4748	0	2	1	21	14	18
							PP4762	4	11	0	32	29	43
PP5242	3	4	3	16	13	9
							PP5656	0	0	0	35	39	32

The cDNA clone is adopted two deoxidation cessation method, on the ABI377 automatic dna sequencer, measure the nucleotide sequence of the nearly 500bp of one end.After the analysis, be defined as novel gene cloning, carry out the other end order-checking.As obtaining full length cDNA sequence not yet, then design primer, check order once more, up to obtaining full length sequence (SEQ ID NO:1,4,7,10,13,16,19,22,24).

Embodiment 2: PCR obtains gene clone from placenta cDNA:

Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (pharmacia company) of purifying.With MMLV-RT-SuperscriptII (GIBCO BRL), ThermoScript II is carried out reverse transcription reaction at 42 ℃, obtains placenta cDNA.Utilize the different primer of commentaries on classics (as shown in the table) of each gene, by 97 ℃ of 3 ' 1 circulation.94 ℃ 30 " 60 ℃ 30 " 72 ℃ 1 ', 35 circulations, pcr amplification is carried out in 72 ℃ of 10 ' 1 circulations, obtains the amplified production of each gene.Amplified production is through sequence verification, and the sequence that records with embodiment 1 conforms to, and changes amplified production over to host cell with routine techniques subsequently, thereby obtains recombinant protein.

The gene specific primer sequence

Clone's title

Special primer 1 (5 ' → 3 ')

Special primer 2 (5 ' → 3 ')

PP4277	TCACGGTCCCACTCAAGTCCTGC	TCTGGACACAGAACGAGCAGCCC
			PP4377	GGCCCTGGGTTCACCTGATTGAG	GAGCCCTCCTCCTAGGGCTGGAA
PP4522	TCCCTGTCCCTGCTTCTCTTCGG	AAGCAGAGGGCGAAAGCTGCAAG
			PP4583	GCGCGTCACCAGAGTCGTTTCTC	GGTCTTTCCACTTCGGGCTTCTG
PP4664	CAACGCGGACTTCATCATCCCTG	CCACCTGATGCAGGCCAACCATA
			PP4748	CGACGCGCTGTCTGGAGTCGTAG	TCTGCTGACATGAGGACCTGGGG
PP4762	TGAATTGGCGCTATGTCTGGGGA	TCAAGCACTTGGCTTCTGCTGCC
			PP5242	GGCTGGGTCCCACCAGTGACAAG	CGACCTGCAGACGGGACATGACT
PP5656	AGAGCTCTTTGTGTGCCCCAGCC	AAGACCCCACCCCACAGATGCTC

Embodiment 3:cDNA cloned sequence is analyzed

1.PP4277 albumen

A: nucleotide sequence (SEQ ID NO:1) length: 1915bp

CACTAAGGCG CAAGGCAGCT GCACATCCAC GGCCAGCCTC GTTGTCTTCA GGGATTTGTT 60

CAAAGTTCAT TCTGAATTGT AAGGTAACCA CTGCAGAACA CGAGCCTTTC CTGGGTATGT 120

GTGGTTATTT CTGCCCTTCA CGGTCCCACT CAAGTCCTGC CTCCCTCAGG GAGACCTCCC 180

AGACCTCACC AAGCCGTGGG CACGTGGACC AGAAGCCGTG GACCCCAGAG GCTCATTTAG 240

GGATCTGCTT TCTGAACCCA GGTCCTAACA TCTGCACTGC AGCGAGCATT TCTGGTGCCT 300

CAAGTGAGTC ACTCCAACTT CCAGCTCACT GAGAGGCGAT GGAGCACTGA GTGCCAGCAT 360

CTTCCAGCAC CAGAGGAGCT GACTCTGAAT AGACAGCTCG GCAGCGGCCT CAGCACAGAG 420

CAGGGGAGCC ACCAGCCACA GGCAGCCACT GAGCCCTTGG CATGTAACCC GGTAACCCAT 480

GCAACAGAAG ACCTGAGCGT CCAGTGTTGC TAGCTTGAAG TAACTTCAGT GGTGTTCCCC 540

AGCGTTTTTC TCATCCCCCT CCCTTCCCTT CCCCTACAGC TTTCTTCCTC TTTCCCCTCC 600

TTCTGAGTCT TTTGTCCCCA GTCCTGGGAA CCTTTTTTCT GACTTGGTGT TTTCTCCTGA 660

ATGCCTCCTT CCTGGCCCCC TAATCAGAAA TGCAGAAGCC ACCTGCCCCA CACAGGGGGC 720

CGCTTTCCCT GCATCTGGCA CTCACCAGGA CAGTCATAGA ACTGGCCTTG GGTGCTTGGG 780

GAGAGTGGAG AGGTCTGGGG GGCATCCACT GGCCTCAGCC TCAGAGCTTC TCATCAGTAA 840

CCCAGGAGGC AAAGGCCCAG CTCGAGATTA GCCCAGGCCA CTGTATCTGT GCCAAGACCT 900

ACAGCTGTCC CTGCAGGGCG TGCATCTCCT GCAGCCCAGG CTATCACAAT GGTGTCCCTT 960

TTGGTGGGGA TGTGAGGCCT GCTGCCTGCC TGGGAAGAAG CTGACAGGAT TGTTGGTGCT 1020

GTGGGGAGAA GCTGGTGTAA CTGAGACCCA TGAGGGAGGC CCTTTGTGGG TCTGTGGCTG 1080

GGAGGAGCTC TGGGGAAGAT GGGCCTTGAG AAATCCCAGC TTAGCACCTT TGACAGTTGC 1140

TCCACTGAGT CACCCTAGAC TGCAATCTCA CAGATGTTCT AACCACCCTC TCCCTGGGGA 1200

GCCCTTGGGG GTGCTGCAAG CCCAGTCCTC ACTCACTGGC TTTGTGCTTC TCTTCCCGAC 1260

TTCCTTTTTT TGCTCCTGTT GAGAAATGTG GGTCCAGGGC TGCGTGCTCC AGTGTGCGGA 1320

AGGAAGGGCA GCGTGCTCTT TGGACTGAGC TGGCTCAAGG GAAGCTGGGT CTGAGGGGAG 1380

ACTGACCTCG ACCCTTCTTA AATGAAACAC CTTTTCCCTG CGTCTCAGTC ACCAGTTACC 1440

TGGCCTGATC ACTGTGTGCC ACACCCCATC ACATCCATCC CCACAAGAAT GCCAGGAGGA 1500

GGGGTCATTG GCCTTTTTGC CAGCACAAAC CTCTGGAGGC CAGCTCCCAA GACCCGCCAG 1560

GCACACCTGC CAGAGTGCCT TAACTTGGGA TGTCTGCAAA CCTCTGTCCA TGGCCGAACC 1620

CACCCCACTG CCTGTTACTG TAAATAAAGG TCTATTAGCA TGCCACCATG CCCCTCCTCC 1680

GATGCATACA GTGCCCAGCT CAGCACCTGG TGCAAAGTAA GCACAATCTG GCTTCCAGCC 1740

ATCACTGCTG TTGCTACTAT CACAAATATT AGGCCCCTTC TAGAATGCAA AGATAACCCA 1800

GGGCTGCTCG TTCTGTGTCC AGATCACAGA GTTTTTCTTT TTATCCTTAT CAACTTTATT 1860

GAGGGATAAT TTACAGGCAA TAAATCATAT CCATTTAAAA AAAAAAAAAA AAAAA 1915

B: aminoacid sequence (SEQ ID NO:2) length: 104 amino acid

1 MQKPPAPHRG PLSLHLALTR TVIELALGAW GEWRGLGGIH WPQPQSFSSV TQEAKAQLEI

61 SPGHCICAKT YSCPCRACIS CSPGYHNGVP FGGDVRPAAC LGRS

C. Nucleotide and amino acid composite sequence (SEQ ID NO:3)

Clone number: PP4277

Start code: 690 ATG stop coding: 1004 TGA

Protein molecular weight: 11076

1 CA CTA AGG CGC AAG GCA GCT GCA CAT CCA CGG CCA GCC TCG TTG TCT 47

48 TCA GGG ATT TGT TCA AAG TTC ATT CTG AAT TGT AAG GTA ACC ACT GCA 95

96 GAA CAC GAG CCT TTC CTG GGT ATG TGT GGT TAT TTC TGC CCT TCA CGG 143

144 TCC CAC TCA AGT CCT GCC TCC CTC AGG GAG ACC TCC CAG ACC TCA CCA 191

192 AGC CGT GGG CAC GTG GAC CAG AAG CCG TGG ACC CCA GAG GCT CAT TTA 239

240 GGG ATC TGC TTT CTG AAC CCA GGT CCT AAC ATC TGC ACT GCA GCG AGC 287

288 ATT TCT GGT GCC TCA AGT GAG TCA CTC CAA CTT CCA GCT CAC TGA GAG 335

336 GCG ATG GAG CAC TGA GTG CCA GCA TCT TCC AGC ACC AGA GGA GCT GAC 383

384 TCT GAA TAG ACA GCT CGG CAG CGG CCT CAG CAC AGA GCA GGG GAG CCA 431

432 CCA GCC ACA GGC AGC CAC TGA GCC CTT GGC ATG TAA CCC GGT AAC CCA 479

480 TGC AAC AGA AGA CCT GAG CGT CCA GTG TTG CTA GCT TGA AGT AAC TTC 527

528 AGT GGT GTT CCC CAG CGT TTT TCT CAT CCC CCT CCC TTC CCT TCC CCT 575

576 ACA GCT TTC TTC CTC TTT CCC CTC CTT CTG AGT CTT TTG TCC CCA GTC 623

624 CTG GGA ACC TTT TTT CTG ACT TGG TGT TTT CTC CTG AAT GCC TCC TTC 671

672 CTG GCC CCC TAA TCA GAA ATG CAG AAG CCA CCT GCC CCA CAC AGG GGG 719

1 Met Gln Lys Pro Pro Ala Pro His Arg Gly 10

720 CCG CTT TCC CTG CAT CTG GCA CTC ACC AGG ACA GTC ATA GAA CTG GCC 767

11 Pro Leu Ser Leu His Leu Ala Leu Thr Arg Thr Val Ile Glu Leu Ala 26

768 TTG GGT GCT TGG GGA GAG TGG AGA GGT CTG GGG GGC ATC CAC TGG CCT 815

27 Leu Gly Ala Trp Gly Glu Trp Arg Gly Leu Gly Gly Ile His Trp Pro 42

816 CAG CCT CAG AGC TTC TCA TCA GTA ACC CAG GAG GCA AAG GCC CAG CTC 863

43 Gln Pro Gln Ser Phe Ser Ser Val Thr Gln Glu Ala Lys Ala Gln Leu 58

864 GAG ATT AGC CCA GGC CAC TGT ATC TGT GCC AAG ACC TAC AGC TGT CCC 911

59 Glu Ile Ser Pro Gly His Cys Ile Cys Ala Lys Thr Tyr Ser Cys Pro 74

912 TGC AGG GCG TGC ATC TCC TGC AGC CCA GGC TAT CAC AAT GGT GTC CCT 959

75 Cys Arg Ala Cys Ile Ser Cys Ser Pro Gly Tyr His Asn Gly Val Pro 90

960 TTT GGT GGG GAT GTG AGG CCT GCT GCC TGC CTG GGA AGA AGC TGA CAG 1007

91 Phe Gly Gly Asp Val Arg Pro Ala Ala Cys Leu Gly Arg Ser *** 105

1008 GAT TGT TGG TGC TGT GGG GAG AAG CTG GTG TAA CTG AGA CCC ATG AGG 1055

1056 GAG GCC CTT TGT GGG TCT GTG GCT GGG AGG AGC TCT GGG GAA GAT GGG 1103

1104 CCT TGA GAA ATC CCA GCT TAG CAC CTT TGA CAG TTG CTC CAC TGA GTC 1151

1152 ACC CTA GAC TGC AAT CTC ACA GAT GTT CTA ACC ACC CTC TCC CTG GGG 1199

1200 AGC CCT TGG GGG TGC TGC AAG CCC AGT CCT CAC TCA CTG GCT TTG TGC 1247

1248 TTC TCT TCC CGA CTT CCT TTT TTT GCT CCT GTT GAG AAA TGT GGG TCC 1295

1296 AGG GCT GCG TGC TCC AGT GTG CGG AAG GAA GGG CAG CGT GCT CTT TGG 1343

1344 ACT GAG CTG GCT CAA GGG AAG CTG GGT CTG AGG GGA GAC TGA CCT CGA 1391

1392 CCC TTC TTA AAT GAA ACA CCT TTT CCC TGC GTC TCA GTC ACC AGT TAC 1439

1440 CTG GCC TGA TCA CTG TGT GCC ACA CCC CAT CAC ATC CAT CCC CAC AAG 1487

1488 AAT GCC AGG AGG AGG GGT CAT TGG CCT TTT TGC CAG CAC AAA CCT CTG 1535

1536 GAG GCC AGC TCC CAA GAC CCG CCA GGC ACA CCT GCC AGA GTG CCT TAA 1583

1584 CTT GGG ATG TCT GCA AAC CTC TGT CCA TGG CCG AAC CCA CCC CAC TGC 1631

1632 CTG TTA CTG TAA ATA AAG GTC TAT TAG CAT GCC ACC ATG CCC CTC CTC 1679

1680 CGA TGC ATA CAG TGC CCA GCT CAG CAC CTG GTG CAA AGT AAG CAC AAT 1727

1728 CTG GCT TCC AGC CAT CAC TGC TGT TGC TAC TAT CAC AAA TAT TAG GCC 1775

1776 CCT TCT AGA ATG CAA AGA TAA CCC AGG GCT GCT CGT TCT GTG TCC AGA 1823

1824 TCA CAG AGT TTT TCT TTT TAT CCT TAT CAA CTT TAT TGA GGG ATA ATT 1871

1872 TAC AGG CAA TAA ATC ATA TCC ATT TAA AAA AAA AAA AAA AAA AA 1915

2.PP4377 albumen

A: nucleotide sequence (SEQ ID NO:4) length: 2132bp

GGACGAGACT TGATGGAGCC ACCTATACAG TGCTGTTCAT TGGCACAGGA GACGGCTGGC 60

TGCTCAAGGC TGTGAGCCTG GGGCCCTGGG TTCACCTGAT TGAGGAGCTG CAGCTGTTTG 120

ACCAGGAGCC CATGAGAAGC CTGGTGCTAT CTCAGAGCAA GAAGCTGCTC TTTGCCGGCT 180

CCCGCTCTCA GCTGGTGCAG CTGCCCGTGG CCGACTGCAT GAAGTATCGC TCCTGTGCAG 240

ACTGTGTCCT CGCCCGGGAC CCCTATTGCG CCTGGAGCGT CAACACCAGC CGCTGTGTGC 300

CATGGGTGGC CACTCTGGAT CTCTACTGAT CCAGCATGTG ATGACCTCGG ACACTTCAGG 360

CATCTGCAAC CTCCGTGGCA GTAAGAAAGT CAGGCCCACT CCCAAAAACA TCACGGTGGT 420

GGCGGGCACA GACCTGGTGC TGCCCTGCCA CCTCTCCTCC AACTTGGCCC ATGCCCGCTG 480

GACCTTTGGG GGCCGGGACC TGCCTGCGGA ACAGCCCGGG TCCTTCCTCT ACGATGCCCG 540

GCTCCAGGCC TGGTTGTGAT GGCTGCCCAG CCCCGCCATG CCGGGGCCTA CCACTGCTTT 600

TCAGAGGAGC AGGGGGCGCG GCTGGCTGCT GAAGGCTACC TTGTGGCTGT CGTGGCAGGC 660

CCTGTCGGTG ACCTTGGAGG CCCGGCCCCC CCTGGAAAAC CTGGGGCTGG TGTGGCTGGC 720

GGTGGTGGCC CTGGGGGCTG TGTGCCTGGT GCTGCTGCTG CTGGTGCTGT CATTGCGCCG 780

GCGGCTGCGG GAAGAGCTGG AGAAAGGGGC CAAGCTACTG AGAGGACCTT GGTGTACCCC 840

CTGGAGCTGC CCAAGGAGCC CACCAGTCCC CCCTTCCGCC CTGTCCTGAA CCAGATGAGA 900

AACTTTGGGA TCCTGTCGTT ACTACTATTC AGATGGCTCC CTTAAGATAG TACCTGGGCA 960

TGCCCGGTGC CAGCCCGGTG GGGGGCCCCC TTCGCCACCT CCAGGCATCC CAGGCCAGCC 1020

TCTGCCTTCT CCAACTCGGC TTCACCTGGG GGGTGGGCGG AACTCAAATG CCAATGGTTA 1080

CGTGCGCTTA CAACTAGGAG GGGAGGACCG GGGAGGGCTC GGGCACCCCC TGCCTGAGCT 1140

CGCGGATGAA CTGAGACGCA AACTGCAGCA ACGCCAGCCA CTGCCCGACT CCAACCCCGA 1200

GGAGTCATCA GTATGAGGGG AACCCCCACC GCGTCGGCGG GAAGCGTGGG AGGTGTAGCT 1260

CCTACTTTTG CACAGGCACC AGCTACCTCA GGGACATGGC ACGGGCACCT GCTCTGTCTG 1320

GGACAGATAC TGCCCAGCAC CCACCCGGCC ATGAGGACCT GCTCTGCTCA GCACGGGCAC 1380

TGACCTTGGT GTGGCTCACC AGGGCACCAG CCTCGCAGAA GGCATCTTCC TCCTCTCTGT 1440

GAATCACAGA CACGCGGGAC CCCAGCCGCC AAAACTTTTC AAGGCAGAAG TTTCAAGATG 1500

TGTGTTTGTC TGTATTTGCA CATGTGTTTG TGTGTGTGTG TATGTGTGTG TCCACGCGCG 1560

TGCGCGCTTG TGGCATAGCC TTCCTGTTTC TGTCAAGTCT TCCCTTGGCC TGGGTCCTCC 1620

TGGTGAGTCA TTGGAGCTAT GAAGGGGAAG GGGTCGTATC ACTTTGTCTC TCCTACCCCC 1680

ACTGCCCCGA GTGTCGGGCA GCGATGTACA TATGGAGGTG GGGTGGACAG GGTGCTGTGC 1740

CCCTTCAGAG GGAGTGCAGG GCTTGGGGTG GGCCTAGTCC TGCTCCTAGG GCTGTGAATG 1800

TTTTCAGGGT GGGGGGAGGG AGATGGAGCC TCCTGTGTGT TTGGGGGGAA GGGTGGGTGG 1860

GGCCTCCCAC TTGGCCCCGG GGTTCAGTGG TATTTTATAC TTGCCTTCTT CCTGTACAGG 1920

GCTGGGAAAA GCTGTGTGAA GGGAGAGAAG GGAAAGGGTG GGCCTGCTGT GGACAATGGC 1980

ATACTCTCTT CCAGCCCTAG GAGGAGGGCT CCTAACAGTG TAACTTATTG TGTCCCCGCG 2040

TATTTATTTG TTGTAAATAT TTGAGTATTT TTATATTGCC AAATAAAATG GGAAAAAAAA 2100

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 2132

B: aminoacid sequence (SEQ ID NO:5) length: 152 amino acid

1 MCVSTRVRAC GIAFLFLSSL PLAWVLLVSH WSYEGEGVVS LCLSYPHCPE CRAAMYIWRW

61 GGQGAVPLQR ECRAWGGPSP APRAVNVFRV GGGRWSLLCV WGEGWVGPPT WPRGSVVFYT

121 CLLPVQGWEK LCEGREGKGW ACCGQWHTLF QP

C. Nucleotide and amino acid composite sequence (SEQ ID NO:6)

Clone number: PP4377

Start code: 1542 ATG stop coding: 2000 TAG

Protein molecular weight: 16914

1 GG ACG AGA CTT GAT GGA GCC ACC TAT ACA GTG CTG TTC ATT GGC ACA 47

48 GGA GAC GGC TGG CTG CTC AAG GCT GTG AGC CTG GGG CCC TGG GTT CAC 95

96 CTG ATT GAG GAG CTG CAG CTG TTT GAC CAG GAG CCC ATG AGA AGC CTG 143

144 GTG CTA TCT CAG AGC AAG AAG CTG CTC TTT GCC GGC TCC CGC TCT CAG 191

192 CTG GTG CAG CTG CCC GTG GCC GAC TGC ATG AAG TAT CGC TCC TGT GCA 239

240 GAC TGT GTC CTC GCC CGG GAC CCC TAT TGC GCC TGG AGC GTC AAC ACC 287

288 AGC CGC TGT GTG CCA TGG GTG GCC ACT CTG GAT CTC TAC TGA TCC AGC 335

336 ATG TGA TGA CCT CGG ACA CTT CAG GCA TCT GCA ACC TCC GTG GCA GTA 383

384 AGA AAG TCA GGC CCA CTC CCA AAA ACA TCA CGG TGG TGG CGG GCA CAG 431

432 ACC TGG TGC TGC CCT GCC ACC TCT CCT CCA ACT TGG CCC ATG CCC GCT 479

480 GGA CCT TTG GGG GCC GGG ACC TGC CTG CGG AAC AGC CCG GGT CCT TCC 527

528 TCT ACG ATG CCC GGC TCC AGG CCT GGT TGT GAT GGC TGC CCA GCC CCG 575

576 CCA TGC CGG GGC CTA CCA CTG CTT TTC AGA GGA GCA GGG GGC GCG GCT 623

624 GGC TGC TGA AGG CTA CCT TGT GGC TGT CGT GGC AGG CCC TGT CGG TGA 671

672 CCT TGG AGG CCC GGC CCC CCC TGG AAA ACC TGG GGC TGG TGT GGC TGG 719

720 CGG TGG TGG CCC TGG GGG CTG TGT GCC TGG TGC TGC TGC TGC TGG TGC 767

768 TGT CAT TGC GCC GGC GGC TGC GGG AAG AGC TGG AGA AAG GGG CCA AGC 815

816 TAC TGA GAG GAC CTT GGT GTA CCC CCT GGA GCT GCC CAA GGA GCC CAC 863

864 CAG TCC CCC CTT CCG CCC TGT CCT GAA CCA GAT GAG AAA CTT TGG GAT 911

912 CCT GTC GTT ACT ACT ATT CAG ATG GCT CCC TTA AGA TAG TAC CTG GGC 959

960 ATG CCC GGT GCC AGC CCG GTG GGG GGC CCC CTT CGC CAC CTC CAG GCA 1007

1008 TCC CAG GCC AGC CTC TGC CTT CTC CAA CTC GGC TTC ACC TGG GGG GTG 1055

1056 GGC GGA ACT CAA ATG CCA ATG GTT ACG TGC GCT TAC AAC TAG GAG GGG 1103

1104 AGG ACC GGG GAG GGC TCG GGC ACC CCC TGC CTG AGC TCG CGG ATG AAC 1151

1152 TGA GAC GCA AAC TGC AGC AAC GCC AGC CAC TGC CCG ACT CCA ACC CCG 1199

1200 AGG AGT CAT CAG TAT GAG GGG AAC CCC CAC CGC GTC GGC GGG AAG CGT 1247

1248 GGG AGG TGT AGC TCC TAC TTT TGC ACA GGC ACC AGC TAC CTC AGG GAC 1295

1296 ATG GCA CGG GCA CCT GCT CTG TCT GGG ACA GAT ACT GCC CAG CAC CCA 1343

1344 CCC GGC CAT GAG GAC CTG CTC TGC TCA GCA CGG GCA CTG ACC TTG GTG 1391

1392 TGG CTC ACC AGG GCA CCA GCC TCG CAG AAG GCA TCT TCC TCC TCT CTG 1439

1440 TGA ATC ACA GAC ACG CGG GAC CCC AGC CGC CAA AAC TTT TCA AGG CAG 1487

1488 AAG TTT CAA GAT GTG TGT TTG TCT GTA TTT GCA CAT GTG TTT GTG TGT 1535

1536 GTG TGT ATG TGT GTG TCC ACG CGC GTG CGC GCT TGT GGC ATA GCC TTC 1583

1 Met Cys Val Ser Thr Arg Val Arg Ala Cys Gly Ile Ala Phe 14

1584 CTG TTT CTG TCA AGT CTT CCC TTG GCC TGG GTC CTC CTG GTG AGT CAT 1631

15 Leu Phe Leu Ser Ser Leu Pro Leu Ala Trp Val Leu Leu Val Ser His 30

1632 TGG AGC TAT GAA GGG GAA GGG GTC GTA TCA CTT TGT CTC TCC TAC CCC 1679

31 Trp Ser Tyr Glu Gly Glu Gly Val Val Ser Leu Cys Leu Ser Tyr Pro 46

1680 CAC TGC CCC GAG TGT CGG GCA GCG ATG TAC ATA TGG AGG TGG GGT GGA 1727

47 His Cys Pro Glu Cys Arg Ala Ala Met Tyr Ile Trp Arg Trp Gly Gly 62

1728 CAG GGT GCT GTG CCC CTT CAG AGG GAG TGC AGG GCT TGG GGT GGG CCT 1775

63 Gln Gly Ala Val Pro Leu Gln Arg Glu Cys Arg Ala Trp Gly Gly Pro 78

1776 AGT CCT GCT CCT AGG GCT GTG AAT GTT TTC AGG GTG GGG GGA GGG AGA 1823

79 Ser Pro Ala Pro Arg Ala Val Asn Val Phe Arg Val Gly Gly Gly Arg 94

1824 TGG AGC CTC CTG TGT GTT TGG GGG GAA GGG TGG GTG GGG CCT CCC ACT 1871

95 Trp Ser Leu Leu Cys Val Trp Gly Glu Gly Trp Val Gly Pro Pro Thr 110

1872 TGG CCC CGG GGT TCA GTG GTA TTT TAT ACT TGC CTT CTT CCT GTA CAG 1919

111 Trp Pro Arg Gly Ser Val Val Phe Tyr Thr Cys Leu Leu Pro Val Gln 126

1920 GGC TGG GAA AAG CTG TGT GAA GGG AGA GAA GGG AAA GGG TGG GCC TGC 1967

127 Gly Trp Glu Lys Leu Cys Glu Gly Arg Glu Gly Lys Gly Trp Ala Cys 142

1968 TGT GGA CAA TGG CAT ACT CTC TTC CAG CCC TAG GAG GAG GGC TCC TAA 2015

143 Cys Gly Gln Trp His Thr Leu Phe Gln Pro *** 153

2016 CAG TGT AAC TTA TTG TGT CCC CGC GTA TTT ATT TGT TGT AAA TAT TTG 2063

2064 AGT ATT TTT ATA TTG CCA AAT AAA ATG GGA AAA AAA AAA AAA AAA AAA 2111

2112 AAA AAA AAA AAA AAA AAA AAA 2132

3.PP4522 albumen

A: nucleotide sequence (SEQ ID NO:7) length: 2035bp

GGCACCTCGA GTTTTTTTTT TTTTTATTCC CAGGACATCA AGGAGACTTT CAATAGGTGT 60

GTTCCCAGTC TTTGCCCTTC GTGACCCAGT GGCATCTGGT TCCCTGTCCC TGCTTCTCTT 120

CGGTATCCCT CTCCTCTCCT TCCTTCCCCA GGACCTGAGT TTCCATCTCC TGGACCCTCC 180

TCTCCTTCCC CTCAGCTTTT GCTTTTCCCT CTGGGAATAT CGTGGTCCCA CCCCCTGCCC 240

GGTCCCCTTC CTCCAGGTGT GAAGAGGTAC AGCTGCAGCC CCCAGAGGTC TGGTCCCCTG 300

ACCCGTGCCA ACCCCATAGC CATGACTTCC TGACAGATGC CATCGTGAGG AAAATGAGCC 360

GGATGTTCTG TCAGGCTGCG AGAGTGGACC TGACGCTGGA CCCTGACACG GCTCACCCGG 420

CCCTGATGCT GTCCCCTGAC CGCCGGGGGG TCCGCCTGGC AGAGCGGCGG CAGGAGGTTG 480

CTGACCATCC CAAGCGCTTC TCGGCCGACT GCTGCGTACT GGGGGCCCAG GGCTTCCGCT 540

CCGGCCGGCA CTACTGGGAG GTAGAGGTGG GCGGGCGGCG GGGCTGGGCG GTGGGTGCTG 600

CCCGTGAATC AACCCATCAT AAGGAAAAGG TGGGCCCTGG GGGTTCCTCC GTGGGCAGCG 660

GGGATGCCAG CTCCTCGCGC CATCACCATC GCCGCCGCCG GCTCCACCTG CCCCAGCAGC 720

CCCTGCTCCA GCGGGAAGTG TGGTGCGTGG GCACCAACGG CAAACGCTAT CAGGCCCAGA 780

GCTCCACAGA ACAGACGCTG CTGAGCCCCA GTGAGAAACC AAGGCGCTTT GGTGTGTACC 840

TGGACTATGA AGCTGGGCGC CTGGGCTTCT ACAACGCAGA GACTCTAGCC CACGTGCACA 900

CCTTCTCGGC TGCCTTCCTG GGCGAGCGTG TCTTTCCTTT CTTCCGGGTG CTCTCCAAGG 960

GCACCCGCAT CAAGCTCTGC CCTTGATTAT CCTGCCACCC GCAGGGGCCC CTCTGTCAGC 1020

ACTTGGGGGG TGGGTGGTGG AGGGTGGCCC GTAAGTTTGA GGGCTCAAAG GCTCTTCCCA 1080

CTGCTTGTTA CTGTGTTGCT TCCCACTCCC CCTTGACCCC AGGCCCCTGC TTCTCCCTCT 1140

AGGAGCCTAA AGAACCCTCC TGGCCTCCAG CTCAGCCTTC TCTCACCTAC TATGTCTGTC 1200

CAACAGGTCT GCATGGGTCC CTGATAATGA GAACAGCTGC CTGGTCTTCT CTCCCAGTCT 1260

GCCTAGCCCA GCCCTGGGAC TGGAATTTGA GTAGGGGATG AGGGGAAATT GTAATTTCAT 1320

TCCTTAACTT CCTTTTCCCC ACCCCTGCTC TTCAACCTCT TTATCAGTTC TGAGGCTGGA 1380

GGGTTTGGGC AAGGCAACAT CCCCATTCCA ATTCCATTTT CTGATGCAGA TTTTAGCTGA 1440

GGGATTTGGA AGCCATTTGG GGAGGCAGGC TGGGCCAAAG GGTAGAGCTG GGTAATAAAT 1500

GTCTATTCTC CTGGGGAGGA GGGATTCTAA ACTTTCCTTC CGTCCTCAAT TTCTACCTCC 1560

ATAGACCGGC CAGAATTTAG CTTCACTTGA GAGAGATCTG GAATGGTCGC CATGATTGAA 1620

ACCACGCACC ATTACATCAT CATTACATTA ATTACATCAA CATAAATTAT TTCTTCCCCC 1680

TTCCCTTTTC CAGCACTCAA CCAAGGAGCA AAGCTCATCC CACCCCACAC CCCTCCCAGG 1740

TCTGCTCACT GCCAGGCTCC TCTCCCCTTT GTTCAGTGGA GCTGGCTTTT CTCCCAGCCC 1800

CTTTCCATGC CTTTCACTCC ATTTGGCAAG CTCTGAGGGG GAGCCTGGGG ACGGGTTTGG 1860

GTCCCCAGGA GGAGAGCCTT GGGTATAATC TATTTTTCTA GGAGCCTCTT GCCTTGTCAC 1920

TTGCAGCTTT CGCCCTCTGC TTTGATGGCT GAGGTGAACT CATGTTCTTT GGGAAAAGGG 1980

AAGGCGTGCT GTGGAAATAA AATGTTTATT TGCTTCTAAA AAAAAAAAAA AAAAA 2035

B: aminoacid sequence (SEQ ID NO:8) length: 210 amino acid

1 MSRMFCQAAR VDLTLDPDTA HPALMLSPDR RGVRLAERRQ EVADHPKRFS ADCCVLGAQG

61 FRSGRHYWEV EVGGRRGWAV GAARESTHHK EKVGPGGSSV GSGDASSSRH HHRRRRLHLP

121 QQPLLQREVW CVGTNGKRYQ AQSSTEQTLL SPSEKPRRFG VYLDYEAGRL GFYNAETLAH

181 VHTFSAAFLG ERVFPFFRVL SKGTRIKLCP

C. Nucleotide and amino acid composite sequence (SEQ ID NO:9)

Clone number: PP4522

Start code: 354 ATG stop coding: 986 TGA

Protein molecular weight: 23607

1 GG CAC CTC GAG TTT TTT TTT TTT TTA TTC CCA GGA CAT CAA GGA GAC 47

48 TTT CAA TAG GTG TGT TCC CAG TCT TTG CCC TTC GTG ACC CAG TGG CAT 95

96 CTG GTT CCC TGT CCC TGC TTC TCT TCG GTA TCC CTC TCC TCT CCT TCC 143

144 TTC CCC AGG ACC TGA GTT TCC ATC TCC TGG ACC CTC CTC TCC TTC CCC 191

192 TCA GCT TTT GCT TTT CCC TCT GGG AAT ATC GTG GTC CCA CCC CCT GCC 239

240 CGG TCC CCT TCC TCC AGG TGT GAA GAG GTA CAG CTG CAG CCC CCA GAG 287

288 GTC TGG TCC CCT GAC CCG TGC CAA CCC CAT AGC CAT GAC TTC CTG ACA 335

336 GAT GCC ATC GTG AGG AAA ATG AGC CGG ATG TTC TGT CAG GCT GCG AGA 383

1 Met Ser Arg Met Phe Cys Gln Ala Ala Arg 10

384 GTG GAC CTG ACG CTG GAC CCT GAC ACG GCT CAC CCG GCC CTG ATG CTG 431

11 Val Asp Leu Thr Leu Asp Pro Asp Thr Ala His Pro Ala Leu Met Leu 26

432 TCC CCT GAC CGC CGG GGG GTC CGC CTG GCA GAG CGG CGG CAG GAG GTT 479

27 Ser Pro Asp Arg Arg Gly Val Arg Leu Ala Glu Arg Arg Gln Glu Val 42

480 GCT GAC CAT CCC AAG CGC TTC TCG GCC GAC TGC TGC GTA CTG GGG GCC 527

43 Ala Asp His Pro Lys Arg Phe Ser Ala Asp Cys Cys Val Leu Gly Ala 58

528 CAG GGC TTC CGC TCC GGC CGG CAC TAC TGG GAG GTA GAG GTG GGC GGG 575

59 Gln Gly Phe Arg Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Gly 74

576 CGG CGG GGC TGG GCG GTG GGT GCT GCC CGT GAA TCA ACC CAT CAT AAG 623

75 Arg Arg Gly Trp Ala Val Gly Ala Ala Arg Glu Ser Thr His His Lys 90

624 GAA AAG GTG GGC CCT GGG GGT TCC TCC GTG GGC AGC GGG GAT GCC AGC 671

91 Glu Lys Val Gly Pro Gly Gly Ser Ser Val Gly Ser Gly Asp Ala Ser 106

672 TCC TCG CGC CAT CAC CAT CGC CGC CGC CGG CTC CAC CTG CCC CAG CAG 719

107 Ser Ser Arg His His His Arg Arg Arg Arg Leu His Leu Pro Gln Gln 122

720 CCC CTG CTC CAG CGG GAA GTG TGG TGC GTG GGC ACC AAC GGC AAA CGC 767

123 Pro Leu Leu Gln Arg Glu Val Trp Cys Val Gly Thr Asn Gly Lys Arg 138

768 TAT CAG GCC CAG AGC TCC ACA GAA CAG ACG CTG CTG AGC CCC AGT GAG 815

139 Tyr Gln Ala Gln Ser Ser Thr Glu Gln Thr Leu Leu Ser Pro Ser Glu 154

816 AAA CCA AGG CGC TTT GGT GTG TAC CTG GAC TAT GAA GCT GGG CGC CTG 863

155 Lys Pro Arg Arg Phe Gly Val Tyr Leu Asp Tyr Glu Ala Gly Arg Leu 170

864 GGC TTC TAC AAC GCA GAG ACT CTA GCC CAC GTG CAC ACC TTC TCG GCT 911

171 Gly Phe Tyr Asn Ala Glu Thr Leu Ala His Val His Thr Phe Ser Ala 186

912 GCC TTC CTG GGC GAG CGT GTC TTT CCT TTC TTC CGG GTG CTC TCC AAG 959

187 Ala Phe Leu Gly Glu Arg Val Phe Pro Phe Phe Arg Val Leu Ser Lys 202

960 GGC ACC CGC ATC AAG CTC TGC CCT TGA TTA TCC TGC CAC CCG CAG GGG 1007

203 Gly Thr Arg Ile Lys Leu Cys Pro *** 211

1008 CCC CTC TGT CAG CAC TTG GGG GGT GGG TGG TGG AGG GTG GCC CGT AAG 1055

1056 TTT GAG GGC TCA AAG GCT CTT CCC ACT GCT TGT TAC TGT GTT GCT TCC 1103

1104 CAC TCC CCC TTG ACC CCA GGC CCC TGC TTC TCC CTC TAG GAG CCT AAA 1151

1152 GAA CCC TCC TGG CCT CCA GCT CAG CCT TCT CTC ACC TAC TAT GTC TGT 1199

1200 CCA ACA GGT CTG CAT GGG TCC CTG ATA ATG AGA ACA GCT GCC TGG TCT 1247

1248 TCT CTC CCA GTC TGC CTA GCC CAG CCC TGG GAC TGG AAT TTG AGT AGG 1295

1296 GGA TGA GGG GAA ATT GTA ATT TCA TTC CTT AAC TTC CTT TTC CCC ACC 1343

1344 CCT GCT CTT CAA CCT CTT TAT CAG TTC TGA GGC TGG AGG GTT TGG GCA 1391

1392 AGG CAA CAT CCC CAT TCC AAT TCC ATT TTC TGA TGC AGA TTT TAG CTG 1439

1440 AGG GAT TTG GAA GCC ATT TGG GGA GGC AGG CTG GGC CAA AGG GTA GAG 1487

1488 CTG GGT AAT AAA TGT CTA TTC TCC TGG GGA GGA GGG ATT CTA AAC TTT 1535

1536 CCT TCC GTC CTC AAT TTC TAC CTC CAT AGA CCG GCC AGA ATT TAG CTT 1583

1584 CAC TTG AGA GAG ATC TGG AAT GGT CGC CAT GAT TGA AAC CAC GCA CCA 1631

1632 TTA CAT CAT CAT TAC ATT AAT TAC ATC AAC ATA AAT TAT TTC TTC CCC 1679

1680 CTT CCC TTT TCC AGC ACT CAA CCA AGG AGC AAA GCT CAT CCC ACC CCA 1727

1728 CAC CCC TCC CAG GTC TGC TCA CTG CCA GGC TCC TCT CCC CTT TGT TCA 1775

1776 GTG GAG CTG GCT TTT CTC CCA GCC CCT TTC CAT GCC TTT CAC TCC ATT 1823

1824 TGG CAA GCT CTG AGG GGG AGC CTG GGG ACG GGT TTG GGT CCC CAG GAG 1871

1872 GAG AGC CTT GGG TAT AAT CTA TTT TTC TAG GAG CCT CTT GCC TTG TCA 1919

1920 CTT GCA GCT TTC GCC CTC TGC TTT GAT GGC TGA GGT GAA CTC ATG TTC 1967

1968 TTT GGG AAA AGG GAA GGC GTG CTG TGG AAA TAA AAT GTT TAT TTG CTT 2015

2016 CTA AAA AAA AAA AAA AAA AA 2035

4.PP4583 albumen

A: nucleotide sequence (SEQ ID NO:10) length: 2142bp

CCGAAACGGG GAAGTTGTCT TGTGTGGAGA GGTTAGTAGA GCAGCGCGCG CGTCACCAGA 60

GTCGTTTCTC TTCGGAGTCT TAGGTGATCG AGGGTGTGCC CAGGGGGCGG GACTTGTTTG 120

CGCCTCCCGT TCCCTCCCAA TTTCCAAACG TGTCACCCCG GCGCCGACGG CCCTGTGCAG 180

GGGAAGCAGA TGGAGTTCAA GCTGGAGGCT CATCGCATCG TCAGCATCTC TCTGGGCAAG 240

ATCTACAACT CGCGGGTCCA GCGCGGCGGC ATCAAGCTGC ATAAGAACCT CCTGGTCTCG 300

CTGGTGCTGC GCAGCGCCCG CCAAGTCTAC CTGAGCGACC CGTGCCCCGG CCTCTACCTG 360

GCCGGTCCCG CTGGGACCCC GGCGCCGCCA CCGCAGCAGC AGCCCGGGGA GCCGGCGGCC 420

GGGCCACCCG CCGGCTGGGG AGAGCCGCCC CCGCCCGCCG CTCATGCCTC TTGGCCGGAG 480

ACCGAGCCGC AGCCGGAGCG CTCCTCCGTC TCAGACGCGC CGCGGGTAGG GGACGAGGTG 540

CCGGTGGCCA CGGTGACTGG AGTCGGGGAC GTTTTTCAGG GCGGAGAGGC GGACGCGACG 600

GAAGCTGCCT GGAGCCGCGT GGAGGGGCCG CGCCAGGCGG CGGCCAGAGA AGCCGAGGGT 660

ACCGCCGGAG GCTGGGGCGT CTTCCCCGAG GTATCTCGTG CCGCGCGCCG CCCCTGCGGC 720

TGCCCCCTAG GCGGGGAGGA CCCGCCGGGT ACACCGGCCG CGACCCCCGG CGCTGCCTGC 780

TGCTGCGCGC CGCGACCAGC GGAGGACGAG CCCCCCGCGC CGCCCGCGGT GTGCCCCAGG 840

AAGCGCTGCG CGGCGGGGGT GGGCGGCGGC CCAGCGGGCT GCCCGGCGCC CGGCTCGACC 900

CCGCTCAAGA AGCCCCGCCG GAACTTAGAG CAGCCGCCGA GTGGAGGAGA GGACGACGAC 960

GCGGAGGAGA TGGAGACCGG GAACGTGGCT AACCTCATCA GCATCTTCGG TTCCAGTTTC 1020

TCGGGACTCC TACGGAAAAG CCCCGGGGGC GGCAGAGAGG AAGAGGAGGG AGAGGAGAGC 1080

GGTCCGGAAG CCGCCGAGCC CGGGCAGATC TGCTGCGATA AGCCGGTGCT GAGAGACATG 1140

AACCCCTGGA GCACAGCCAT CGTGGCCTTC TGAGCCCTTG GCCCCCCTGC GGGGAGGAGG 1200

TGGAGCAGCG GGCGTCCCCG AAGTGAGGGC CAGGCCCTTC CCGGCTGCGA GGACGCCCAG 1260

AGACCGCGGG CGCTGAGCGC GTTGGGCAGA CTGGTTGCCT CTGGGCATCG CAAACTGCCC 1320

CCGGGCGCAT CTGGCTTATC TGGAGACCTG GGAGCTTGAG GCTCATTGGA AGAGGACGAT 1380

CGTTAACTTT TGTGTTTGTG TATGTCTGTG TATGTAAGTT TGTCTTGTGG CATTAAGACT 1440

ATTTTGCTCT TCTGGAATGC ACCACTCCTT CCCCAGGGTT TAAGAGATTG GGGGCAGACA 1500

GGGACTTTCC TCTGCCGGCT GCGTGGAGAA GGAAGCGGCT AAAAGGTTTG GGCCGGGGAG 1560

TTCCCCATTC GTTCTCCGGG GAGGGAGGGA CTTTACACCT ACCCCTCACC GGAAAGCTAG 1620

ACCCGCTTCA GGGCCAGGAG TGGCGTTTCC GCACAGGATT TCCTAAGACG AGAGGGATTT 1680

AGCCAAGAGC ACAGACTTGG ATTCCTTCTG TCCCTCCCCA CCTTTCTCTC CCAATGAAGA 1740

AACGTTTATA TCCTGTGATT ACTCTAGGAA AGGGGCGTAT TAACGGTCCT TTGCTTCCCC 1800

GCAGGGGAGA AAAAGCTTGA CGAACTTCAC AGAAGAGTTC AAGCTTGGAA ATAATGGAGT 1860

TTATAGAGAA AAGATATATT TTTAAAAGCT CTAGGTCAGA AGTACTACAC AGTGCTTTTA 1920

AAAAGTGTTT AATGAGAGTT TACAGACAGA AGCCCGAAGT GGAAAGACCT TATGGTTTTG 1980

TAGATATGGT GACTCCAGTC TTTGTTGTAT AAAGGTTGGG GGAGCTGATA AGGTTTTTGT 2040

ACAGTATTTT CTCCTTCGTT GTATTGATTT TTGTATAAAA ATGTAACTTC TACGTGTCTA 2100

ACACGTATTA AATATTTTGA AGCAAAAAAA AAAAAAAAAA AA 2142

B: aminoacid sequence (SEQ ID NO:11) length: 327 amino acid

1 MEFKLEAHRI VSISLGKIYN SRVQRGGIKL HKNLLVSLVL RSARQVYLSD PCPGLYLAGP

61 AGTPAPPPQQ QPGEPAAGPP AGWGEPPPPA AHASWPETEP QPERSSVSDA PRVGDEVPVA

121 TVTGVGDVFQ GGEADATEAA WSRVEGPRQA AAREAEGTAG GWGVFPEVSR AARRPCGCPL

181 GGEDPPGTPA ATPGAACCCA PRPAEDEPPA PPAVCPRKRC AAGVGGGPAG CPAPGSTPLK

241 KPRRNLEQPP SGGEDDDAEE METGNVANLI SIFGSSFSGL LRKSPGGGRE EEEGEESGPE

301 AAEPGQICCD KPVLRDMNPW STAIVAF

C. Nucleotide and amino acid composite sequence (SEQ ID NO:12)

Clone number: PP4583

Start code: 190 ATG stop coding: 1173 TGA

Protein molecular weight: 33612

1 CCG AAA CGG GGA AGT TGT CTT GTG TGG AGA GGT TAG TAG AGC AGC GCG 48

49 CGC GTC ACC AGA GTC GTT TCT CTT CGG AGT CTT AGG TGA TCG AGG GTG 96

97 TGC CCA GGG GGC GGG ACT TGT TTG CGC CTC CCG TTC CCT CCC AAT TTC 144

145 CAA ACG TGT CAC CCC GGC GCC GAC GGC CCT GTG CAG GGG AAG CAG ATG 192

1 Met 1

193 GAG TTC AAG CTG GAG GCT CAT CGC ATC GTC AGC ATC TCT CTG GGC AAG 240

2 Glu Phe Lys Leu Glu Ala His Arg Ile Val Ser Ile Ser Leu Gly Lys 17

241 ATC TAC AAC TCG CGG GTC CAG CGC GGC GGC ATC AAG CTG CAT AAG AAC 288

18 Ile Tyr Asn Ser Arg Val Gln Arg Gly Gly Ile Lys Leu His Lys Asn 33

289 CTC CTG GTC TCG CTG GTG CTG CGC AGC GCC CGC CAA GTC TAC CTG AGC 336

34 Leu Leu Val Ser Leu Val Leu Arg Ser Ala Arg Gln Val Tyr Leu Ser 49

337 GAC CCG TGC CCC GGC CTC TAC CTG GCC GGT CCC GCT GGG ACC CCG GCG 384

50 Asp Pro Cys Pro Gly Leu Tyr Leu Ala Gly Pro Ala Gly Thr Pro Ala 65

385 CCG CCA CCG CAG CAG CAG CCC GGG GAG CCG GCG GCC GGG CCA CCC GCC 432

66 Pro Pro Pro Gln Gln Gln Pro Gly Glu Pro Ala Ala Gly Pro Pro Ala 81

433 GGC TGG GGA GAG CCG CCC CCG CCC GCC GCT CAT GCC TCT TGG CCG GAG 480

82 Gly Trp Gly Glu Pro Pro Pro Pro Ala Ala His Ala Ser Trp Pro Glu 97

481 ACC GAG CCG CAG CCG GAG CGC TCC TCC GTC TCA GAC GCG CCG CGG GTA 528

98 Thr Glu Pro Gln Pro Glu Arg Ser Ser Val Ser Asp Ala Pro Arg Val 113

529 GGG GAC GAG GTG CCG GTG GCC ACG GTG ACT GGA GTC GGG GAC GTT TTT 576

114 Gly Asp Glu Val Pro Val Ala Thr Val Thr Gly Val Gly Asp Val Phe 129

577 CAG GGC GGA GAG GCG GAC GCG ACG GAA GCT GCC TGG AGC CGC GTG GAG 624

130 Gln Gly Gly Glu Ala Asp Ala Thr Glu Ala Ala Trp Ser Arg Val Glu 145

625 GGG CCG CGC CAG GCG GCG GCC AGA GAA GCC GAG GGT ACC GCC GGA GGC 672

146 Gly Pro Arg Gln Ala Ala Ala Arg Glu Ala Glu Gly Thr Ala Gly Gly 161

673 TGG GGC GTC TTC CCC GAG GTA TCT CGT GCC GCG CGC CGC CCC TGC GGC 720

162 Trp Gly Val Phe Pro Glu Val Ser Arg Ala Ala Arg Arg Pro Cys Gly 177

721 TGC CCC CTA GGC GGG GAG GAC CCG CCG GGT ACA CCG GCC GCG ACC CCC 768

178 Cys Pro Leu Gly Gly Glu Asp Pro Pro Gly Thr Pro Ala Ala Thr Pro 193

769 GGC GCT GCC TGC TGC TGC GCG CCG CGA CCA GCG GAG GAC GAG CCC CCC 816

194 Gly Ala Ala Cys Cys Cys Ala Pro Arg Pro Ala Glu Asp Glu Pro Pro 209

817 GCG CCG CCC GCG GTG TGC CCC AGG AAG CGC TGC GCG GCG GGG GTG GGC 864

210 Ala Pro Pro Ala Val Cys Pro Arg Lys Arg Cys Ala Ala Gly Val Gly 225

865 GGC GGC CCA GCG GGC TGC CCG GCG CCC GGC TCG ACC CCG CTC AAG AAG 912

226 Gly Gly Pro Ala Gly Cys Pro Ala Pro Gly Ser Thr Pro Leu Lys Lys 241

913 CCC CGC CGG AAC TTA GAG CAG CCG CCG AGT GGA GGA GAG GAC GAC GAC 960

242 Pro Arg Arg Asn Leu Glu Gln Pro Pro Ser Gly Gly Glu Asp Asp Asp 257

961 GCG GAG GAG ATG GAG ACC GGG AAC GTG GCT AAC CTC ATC AGC ATC TTC 1008

258 Ala Glu Glu Met Glu Thr Gly Asn Val Ala Asn Leu Ile Ser Ile Phe 273

1009 GGT TCC AGT TTC TCG GGA CTC CTA CGG AAA AGC CCC GGG GGC GGC AGA 1056

274 Gly Ser Ser Phe Ser Gly Leu Leu Arg Lys Ser Pro Gly Gly Gly Arg 289

1057 GAG GAA GAG GAG GGA GAG GAG AGC GGT CCG GAA GCC GCC GAG CCC GGG 1104

290 Glu Glu Glu Glu Gly Glu Glu Ser Gly Pro Glu Ala Ala Glu Pro Gly 305

1105 GAG ATC TGC TGC GAT AAG CCG GTG CTG AGA GAC ATG AAC CCC TGG AGC 1152

306 Gln Ile Cys Cys Asp Lys Pro Val Leu Arg Asp Met Asn Pro Trp Ser 321

1153 ACA GCC ATC GTG GCC TTC TGA GCC CTT GGC CCC CCT GCG GGG AGG AGG 1200

322 Thr Ala Ile Val Ala Phe *** 328

1201 TGG AGC AGC GGG CGT CCC CGA AGT GAG GGC CAG GCC CTT CCC GGC TGC 1248

1249 GAG GAC GCC CAG AGA CCG CGG GCG CTG AGC GCG TTG GGC AGA CTG GTT 1296

1297 GCC TCT GGG CAT CGC AAA CTG CCC CCG GGC GCA TCT GGC TTA TCT GGA 1344

1345 GAC CTG GGA GCT TGA GGC TCA TTG GAA GAG GAC GAT CGT TAA CTT TTG 1392

1393 TGT TTG TGT ATG TCT GTG TAT GTA AGT TTG TCT TGT GGC ATT AAG ACT 1440

1441 ATT TTG CTC TTC TGG AAT GCA CCA CTC CTT CCC CAG GGT TTA AGA GAT 1488

1489 TGG GGG CAG ACA GGG ACT TTC CTC TGC CGG CTG CGT GGA GAA GGA AGC 1536

1537 GGC TAA AAG GTT TGG GCC GGG GAG TTC CCC ATT CGT TCT CCG GGG AGG 1584

1585 GAG GGA CTT TAC ACC TAC CCC TCA CCG GAA AGC TAG ACC CGC TTC AGG 1632

1633 GCC AGG AGT GGC GTT TCC GCA CAG GAT TTC CTA AGA CGA GAG GGA TTT 1680

1681 AGC CAA GAG CAC AGA CTT GGA TTC CTT CTG TCC CTC CCC ACC TTT CTC 1728

1729 TCC CAA TGA AGA AAC GTT TAT ATC CTG TGA TTA CTC TAG GAA AGG GGC 1776

1777 GTA TTA ACG GTC CTT TGC TTC CCC GCA GGG GAG AAA AAG CTT GAC GAA 1824

1825 CTT CAC AGA AGA GTT CAA GCT TGG AAA TAA TGG AGT TTA TAG AGA AAA 1872

1873 GAT ATA TTT TTA AAA GCT CTA GGT CAG AAG TAC TAC ACA GTG CTT TTA 1920

1921 AAA AGT GTT TAA TGA GAG TTT ACA GAC AGA AGC CCG AAG TGG AAA GAC 1968

1969 CTT ATG GTT TTG TAG ATA TGG TGA CTC CAG TCT TTG TTG TAT AAA GGT 2016

2017 TGG GGG AGC TGA TAA GGT TTT TGT ACA GTA TTT TCT CCT TCG TTG TAT 2064

2065 TGA TTT TTG TAT AAA AAT GTA ACT TCT ACG TGT CTA ACA CGT ATT AAA 2112

2113 TAT TTT GAA GCA AAA AAA AAA AAA AAA AAA 2142

5.PP4664 albumen

A: nucleotide sequence (SEQ ID NO:13) length: 2158bp

GCCATCCCTA AGCAGACCCC AGTCCAATAC CTGCTCCCTG AGGCCAAGGC CCAGGACTCA 60

GACAAGATCT GCGTGGTCAT CGACCTGGAC GAGACCCTGG TGCACAGCTC CTTCAAGCCA 120

GTGAACAACG CGGACTTCAT CATCCCTGTG GAGATTGATG GGGTGGTCCA CCAGGTCTAC 180

GTGTTGAAGC GTCCTCATGT GGATGAGTTC CTGCAGCGAA TGGGCGAGCT CTTTGAATGT 240

GTGCTGTTCA CTGCTAGCCT CGCCAAGTAC GCAGACCCAG TAGCTGACCT GCTGGACAAA 300

TGGGGGGCCT TCCGGGCCCG GCTGTTTCGA GAGTCCTGCG TCTTCCACCG GGGGAACTAC 360

GTGAAGGACC TGAGCCGGTT GGGTCGAGAC CTGCGGCGGG TGCTCATCCT GGACAATTCA 420

CCTGCCTCCT ATGTCTTCCA TCCAGACAAT GCTGTACCGG TGGCCTCGTG GTTTGACAAC 480

ATGAGTGACA CAGAGCTCCA CGACCTCCTC CCCTTCTTCG AGCAACTCAG CCGTGTGGAC 540

GACGTGTACT CAGTGCTCAG GCAGCCACGG CCAGGGAGCT AGTGAGGGTG ATGGGGCCAG 600

GACCTGCCCC TGACCAATGA TACCCACACC TCCTCCCAGG AAGACTGCCC AGGCCTTTGT 660

TAGGAAAACC CATGGGCCGC CGCCACACTC AGTGCCATGG GGAAGCGGGC GTCTCCCCCA 720

CCAGCCCCAC CAGGCGGTGT AGGGGCAGCA GGCTGCACTG AGGACCGTGA GCTCCAGGCC 780

CCGTGTCAGT GCCTTCAAAC CTCCTCCCCT ATTCTCAGGG GACCTGGGGG GCCCTGCCTG 840

CTGCTCCCTT TTTCTGTCTC TGTCCATGCT GCCATGTTTC TCTGCTGCCA AATTGGGCCC 900

CTTGGCCCCT TCCGGTTCTG CTTCCTGGGG GCAGGGTTCC TGCCTTGGAC CCCCAGTCTG 960

GGAACGGTGG ACATCAAGTG CCTTGCATAG AGCCCCCTCT TCCCCGCCCA GCTTTCCCAG 1020

GGGCACAGCT CTAGGCTGGG AGGGGAGAAC CAGCCCCTCC CCCTGCCCCA CCTCCTCCCT 1080

TGGGACTGAG AGGGCCCCTA CCAACCTTTG CCTCTGCCTT GGAGGGAGGG GAGGTCTGTT 1140

ACCACTGGGG AAGGCAGCAG GAGTCTGTCC TTCAGGCCCC ACAGTGCAGC TTCTCCAGGG 1200

CCGACAGCTG AGGGCTGCTC CCTGCATCAT CCAAGCAATG ACCTCAGACT TCTGCCTTAA 1260

CCAGCCCCGG GGCTTGGCTC CCCCAGCTCT GAGCGTGGGG GCATAGGCAG GACCCCCCTT 1320

GTGGTGCCAT ATAAATATGT ACATGTGTAT ATAGATTTTT AGGGGAAGGA GAGAGGGAAG 1380

GGTCAGGGTA GAGACACCCC TCCCTTGCCC CTTTCCTGGG CCCAGAAGTT GGGGGGAGGG 1440

AGGGAAAGGA TTTTTACATT TTTTAAACTG CTATTTTCTG AATGGAACAA GCTGGGCCAA 1500

GGGGCCCAGG CCCTGTCCTC TGTCCCTCAC ACCCCTTTGC TCCGTTCATT CATTCAAAAA 1560

AACATTTCTT GAGCACCTTC TGTGCCCAGC ATATGCTAGG CCCACCAGCT AAGTGTGTGT 1620

GGGGGGTCTC TACGCCAGCT CATCAGTGCC TCCTTGCCCA TCCTTCACCG GTGCCTTTGG 1680

GGGATCTGTA GGAGGTGGGA CCTTCTGTGG GGTTTGGGGA TCTCCAGGAA GCCCGACCAA 1740

GCTGTCCCCT TCCCCTGTGC CAACCCATCT CCTACAGCCC CCTGCCTGAT CCCCTGCTGG 1800

CTGGGGGCAG CTCCCAGGAT ATCCTGCCTT CCAACTGTTT CTGAAGCCCC TCCTCCTAAC 1860

ATGGCGATTC CGGAGGTCAA GGCCTTGGGC TCTCCCCAGG GTCTAACGGT TAAGGGGACC 1920

CACATACCAG TGCCAAGGGG GATGTCAAGT GGTGATGTCG TTGTGCTCCC CTCCCCCAGA 1980

GCGGGTGGGC GGGGGGTGAA TATGGTTGGC CTGCATCAGG TGGCCTTCCC ATTTAAGTGC 2040

CTTCTCTGTG ACTGAGAGCC CTAGTGTGAT GAGAACTAAA GAGAAAGCCA GACCCCTAAA 2100

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 2158

B: aminoacid sequence (SEQ ID NO:14) length: 146 amino acid

1 MLPCFSAAKL GPLAPSGSAS WGQGSCLGPP VWERWTSSAL HRAPSSPPSF PRGTALGWEG

61 RTSPSPCPTS SLGTERAPTN LCLCLGGRGG LLPLGKAAGV CPSGPTVQLL QGRQLRAAPC

121 IIQAMTSDFC LNQPRGLAPP ALSVGA

C. Nucleotide and amino acid composite sequence (SEQ ID NO:15)

Clone number: PP4664

Start code: 866 ATG stop coding: 1306 TAG

Protein molecular weight: 14815

1 G CCA TCC CTA AGC AGA CCC CAG TCC AAT ACC TGC TCC CTG AGG CCA 46

47 AGG CCC AGG ACT CAG ACA AGA TCT GCG TGG TCA TCG ACC TGG ACG AGA 94

95 CCC TGG TGC ACA GCT CCT TCA AGC CAG TGA ACA ACG CGG ACT TCA TCA 142

143 TCC CTG TGG AGA TTG ATG GGG TGG TCC ACC AGG TCT ACG TGT TGA AGC 190

191 GTC CTC ATG TGG ATG AGT TCC TGC AGC GAA TGG GCG AGC TCT TTG AAT 238

239 GTG TGC TGT TCA CTG CTA GCC TCG CCA AGT ACG CAG ACC CAG TAG CTG 286

287 ACC TGC TGG ACA AAT GGG GGG CCT TCC GGG CCC GGC TGT TTC GAG AGT 334

335 CCT GCG TCT TCC ACC GGG GGA ACT ACG TGA AGG ACC TGA GCC GGT TGG 382

383 GTC GAG ACC TGC GGC GGG TGC TCA TCC TGG ACA ATT CAC CTG CCT CCT 430

431 ATG TCT TCC ATC CAG ACA ATG CTG TAC CGG TGG CCT CGT GGT TTG ACA 478

479 ACA TGA GTG ACA CAG AGC TCC ACG ACC TCC TCC CCT TCT TCG AGC AAC 526

527 TCA GCC GTG TGG ACG ACG TGT ACT CAG TGC TCA GGC AGC CAC GGC CAG 574

575 GGA GCT AGT GAG GGT GAT GGG GCC AGG ACC TGC CCC TGA CCA ATG ATA 622

623 CCC ACA CCT CCT CCC AGG AAG ACT GCC CAG GCC TTT GTT AGG AAA ACC 670

671 CAT GGG CCG CCG CCA CAC TCA GTG CCA TGG GGA AGC GGG CGT CTC CCC 718

719 CAC CAG CCC CAC CAG GCG GTG TAG GGG CAG CAG GCT GCA CTG AGG ACC 766

767 GTG AGC TCC AGG CCC CGT GTC AGT GCC TTC AAA CCT CCT CCC CTA TTC 814

815 TCA GGG GAC CTG GGG GGC CCT GCC TGC TGC TCC CTT TTT CTG TCT CTG 862

863 TCC ATG CTG CCA TGT TTC TCT GCT GCC AAA TTG GGC CCC TTG GCC CCT 910

1 Met Leu Pro Cys Phe Ser Ala Ala Lys Leu Gly Pro Leu Ala Pro 15

911 TCC GGT TCT GCT TCC TGG GGG CAG GGT TCC TGC CTT GGA CCC CCA GTC 958

16 Ser Gly Ser Ala Ser Trp Gly Gln Gly Ser Cys Leu Gly Pro Pro Val 31

959 TGG GAA CGG TGG ACA TCA AGT GCC TTG CAT AGA GCC CCC TCT TCC CCG 1006

32 Trp Glu Arg Trp Thr Ser Ser Ala Leu His Arg Ala Pro Ser Ser Pro 47

1007 CCC AGC TTT CCC AGG GGC ACA GCT CTA GGC TGG GAG GGG AGA ACC AGC 1054

48 Pro Ser Phe Pro Arg Gly Thr Ala Leu Gly Trp Glu Gly Arg Thr Ser 63

1055 CCC TCC CCC TGC CCC ACC TCC TCC CTT GGG ACT GAG AGG GCC CCT ACC 1102

64 Pro Ser Pro Cys Pro Thr Ser Ser Leu Gly Thr Glu Arg Ala Pro Thr 79

1103 AAC CTT TGC CTC TGC CTT GGA GGG AGG GGA GGT CTG TTA CCA CTG GGG 1150

80 Asn Leu Cys Leu Cys Leu Gly Gly Arg Gly Gly Leu Leu Pro Leu Gly 95

1151 AAG GCA GCA GGA GTC TGT CCT TCA GGC CCC ACA GTG CAG CTT CTC CAG 1198

96 Lys Ala Ala Gly Val Cys Pro Ser Gly Pro Thr Val Gln Leu Leu Gln 111

1199 GGC CGA CAG CTG AGG GCT GCT CCC TGC ATC ATC CAA GCA ATG ACC TCA 1246

112 Gly Arg Gln Leu Arg Ala Ala Pro Cys Ile Ile Gln Ala Met Thr Ser 127

1247 GAC TTC TGC CTT AAC CAG CCC CGG GGC TTG GCT CCC CCA GCT CTG AGC 1294

128 Asp Phe Cys Leu Asn Gln Pro Arg Gly Leu Ala Pro Pro Ala Leu Ser 143

1295 GTG GGG GCA TAG GCA GGA CCC CCC TTG TGG TGC CAT ATA AAT ATG TAC 1342

144 Val Gly Ala *** 147

1343 ATG TGT ATA TAG ATT TTT AGG GGA AGG AGA GAG GGA AGG GTC AGG GTA 1390

1391 GAG ACA CCC CTC CCT TGC CCC TTT CCT GGG CCC AGA AGT TGG GGG GAG 1438

1439 GGA GGG AAA GGA TTT TTA CAT TTT TTA AAC TGC TAT TTT CTG AAT GGA 1486

1487 ACA AGC TGG GCC AAG GGG CCC AGG CCC TGT CCT CTG TCC CTC ACA CCC 1534

1535 CTT TGC TCC GTT CAT TCA TTC AAA AAA ACA TTT CTT GAG CAC CTT CTG 1582

1583 TGC CCA GCA TAT GCT AGG CCC ACC AGC TAA GTG TGT GTG GGG GGT CTC 1630

1631 TAC GCC AGC TCA TCA GTG CCT CCT TGC CCA TCC TTC ACC GGT GCC TTT 1678

1679 GGG GGA TCT GTA GGA GGT GGG ACC TTC TGT GGG GTT TGG GGA TCT CCA 1726

1727 GGA AGC CCG ACC AAG CTG TCC CCT TCC CCT GTG CCA ACC CAT CTC CTA 1774

1775 CAG CCC CCT GCC TGA TCC CCT GCT GGC TGG GGG CAG CTC CCA GGA TAT 1822

1823 CCT GCC TTC CAA CTG TTT CTG AAG CCC CTC CTC CTA ACA TGG CGA TTC 1870

1871 CGG AGG TCA AGG CCT TGG GCT CTC CCC AGG GTC TAA CGG TTA AGG GGA 1918

1919 CCC ACA TAC CAG TGC CAA GGG GGA TGT CAA GTG GTG ATG TCG TTG TGC 1966

1967 TCC CCT CCC CCA GAG CGG GTG GGC GGG GGG TGA ATA TGG TTG GCC TGC 2014

2015 ATC AGG TGG CCT TCC CAT TTA AGT GCC TTC TCT GTG ACT GAG AGC CCT 2062

2063 AGT GTG ATG AGA ACT AAA GAG AAA GCC AGA CCC CTA AAA AAA AAA AAA 2110

2111 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2158

6.PP4748 albumen

A: nucleotide sequence (SEQ ID NO:16) length: 1337bp

GAACAGCGGG GGCACACGGG GCGCTGCCGA AGTGCAAGGC CACGGCCAGA GCTCGAGCCC 60

GACGCGCTGT CTGGAGTCGT AGGTTGGCGC CGTTTGGGGT CGGGGTCTGA GGCTTGGGCG 120

CTGCCTGGGC CGAGCGGAGA TCGGGGTTTG CCTCCCGTCC CCGCTCAGGA CCCTGACGTG 180

GCTGAAGCGG CCCCGGGAGC ATGAGCGGGC AGCGCGTGGA CGTCAAGGTG GTGATGCTGG 240

GCAAGGAGTA CGTGGGCAAG ACTAGCCTGG TGGAGCGCTA CGTGCACGAC CGCTTTCTGG 300

TGGGGCCTTA TCAGAACACC ATCGGGGCCG CCTTCGTGGC CAAGGTGATG TCGGTCGGAG 360

ACCGGACTGT GACATTAGGT ATTTGGGACA CAGCAGGCTC TGAGCGCTAT GAGGCCATGA 420

GTAGAATCTA CTATCGGGGT GCCAAGGCTG CCATCGTCTG CTATGACCTC ACAGACAGCA 480

GCAGCTTTGA GCGAGCAAAG TTCTGGGTGA AGGAACTGCG CAGCCTAGAG GAGGGCTGCC 540

AAATCTACTT ATGTGGCACC AAGAGTGACC TGCTGGAAGA AGACCGGAGG CGTCGACGTG 600

TGGACTTCCA CGACGTCCAG GACTATGCAG ACAGTAGCTG CTCCTCAGCC CTTTGGGGGG 660

TGGGGGTGTG TGGCTGTCTG GGTGGATCAA AGAAAATAGG GACTGCCTTG GCTGCCAGGG 720

CAAGGTGCTC TAGGAGGTCT TCCTGGCCTC CTTGAACTGT GGGGTCCAGG AGACTCCCTG 780

AACTGCTAGC CCTCCCTTTT GTCTGTTTAT CTAATTCTCA GGTATGAGGC TTTAGTCACT 840

TCTCTTTACA GATATCAAAG CTCAGCTCTT TGAAACATCC AGCAAGACAG GCCAGAGTGT 900

GGACGAGCTC TTCCAGAAAG TGGCAGAGGA TTACGTCAGT GTGGCTGCCT TCCAGGTGAT 960

GACAGAGGAC AAGGGCGTGG ATCTGGGCCA GAAGCCAAAC CCCTACTTCT ACAGCTGTTG 1020

TCATCACTGA GTCAGCACTC ACCTGGCCTG GGGGAATTAA AGGAATTCCC CGTAAGGGCT 1080

GGACCCAGCT CCTTTCTGGG CTTGGGTAGT CAAATGTCTG AGCTACCCCA GGTCCTCATG 1140

TCAGCAGAGT GGCGCCTGCC TGTGCTGGCC CATGGAACGG AGACAGCATT GGGCTGACTG 1200

TGGGCATGAG GAGGGATAAG GCTGATTTGG ACCCCAGGCT TCTGCCCTGG ACAGCACTTG 1260

TGTCTGCAGA TTATTTAAGT GGCTTTTGAT CTGTAAATAA AATCAGTGCA CTGTGCATCA 1320

AAAAAAAAAA AAAAAAA 1337

B: aminoacid sequence (SEQ ID NO:17) length: 184 amino acid

1 MSGQRVDVKV VMLGKEYVGK TSLVERYVHD RFLVGPYQNT IGAAFVAKVM SVGDRTVTLG

61 IWDTAGSERY EAMSRIYYRG AKAAIVCYDL TDSSSFERAK FWVKELRSLE EGCQIYLCGT

121 KSDLLEEDRR RRRVDFHDVQ DYADSSCSSA LWGVGVCGCL GGSKKIGTAL AARARCSRRS

181 SWPP

C. Nucleotide and amino acid composite sequence (SEQ ID NO:18)

Clone number: PP4748

Start code: 201 ATG stop coding: 755 TGA

Protein molecular weight: 20517

1 GA ACA GCG GGG GCA CAC GGG GCG CTG CCG AAG TGC AAG GCC ACG GCC 47

48 AGA GCT CGA GCC CGA CGC GCT GTC TGG AGT CGT AGG TTG GCG CCG TTT 95

96 GGG GTC GGG GTC TGA GGC TTG GGC GCT GCC TGG GCC GAG CGG AGA TCG 143

144 GGG TTT GCC TCC CGT CCC CGC TCA GGA CCC TGA CGT GGC TGA AGC GGC 191

192 CCC GGG AGC ATG AGC GGG CAG CGC GTG GAC GTC AAG GTG GTG ATG CTG 239

1 Met Ser Gly Gln Arg Val Asp Val Lys Val Val Met Leu 13

240 GGC AAG GAG TAC GTG GGC AAG ACT AGC CTG GTG GAG CGC TAC GTG CAC 287

14 Gly Lys Glu Tyr Val Gly Lys Thr Ser Leu Val Glu Arg Tyr Val His 29

288 GAC CGC TTT CTG GTG GGG CCT TAT CAG AAC ACC ATC GGG GCC GCC TTC 335

30 Asp Arg Phe Leu Val Gly Pro Tyr Gln Asn Thr Ile Gly Ala Ala Phe 45

336 GTG GCC AAG GTG ATG TCG GTC GGA GAC CGG ACT GTG ACA TTA GGT ATT 383

46 Val Ala Lys Val Met Ser Val Gly Asp Arg Thr Val Thr Leu Gly Ile 61

384 TGG GAC ACA GCA GGC TCT GAG CGC TAT GAG GCC ATG AGT AGA ATC TAC 431

62 Trp Asp Thr Ala Gly Ser Glu Arg Tyr Glu Ala Met Ser Arg Ile Tyr 77

432 TAT CGG GGT GCC AAG GCT GCC ATC GTC TGC TAT GAC CTC ACA GAC AGC 479

78 Tyr Arg Gly Ala Lys Ala Ala Ile Val Cys Tyr Asp Leu Thr Asp Ser 93

480 AGC AGC TTT GAG CGA GCA AAG TTC TGG GTG AAG GAA CTG CGC AGC CTA 527

94 Ser Ser Phe Glu Arg Ala Lys Phe Trp Val Lys Glu Leu Arg Ser Leu 109

528 GAG GAG GGC TGC CAA ATC TAC TTA TGT GGC ACC AAG AGT GAC CTG CTG 575

110 Glu Glu Gly Cys Gln Ile Tyr Leu Cys Gly Thr Lys Ser Asp Leu Leu 125

576 GAA GAA GAC CGG AGG CGT CGA CGT GTG GAC TTC CAC GAC GTC CAG GAC 623

126 Glu Glu Asp Arg Arg Arg Arg Arg Val Asp Phe His Asp Val Gln Asp 141

624 TAT GCA GAC AGT AGC TGC TCC TCA GCC CTT TGG GGG GTG GGG GTG TGT 671

142 Tyr Ala Asp Ser Ser Cys Ser Ser Ala Leu Trp Gly Val Gly Val Cys 157

672 GGC TGT CTG GGT GGA TCA AAG AAA ATA GGG ACT GCC TTG GCT GCC AGG 719

158 Gly Cys Leu Gly Gly Ser Lys Lys Ile Gly Thr Ala Leu Ala Ala Arg 173

720 GCA AGG TGC TCT AGG AGG TCT TCC TGG CCT CCT TGA ACT GTG GGG TCC 767

174 Ala Arg Cys Ser Arg Arg Ser Ser Trp Pro Pro *** 185

768 AGG AGA CTC CCT GAA CTG CTA GCC CTC CCT TTT GTC TGT TTA TCT AAT 815

816 TCT CAG GTA TGA GGC TTT AGT CAC TTC TCT TTA CAG ATA TCA AAG CTC 863

864 AGC TCT TTG AAA CAT CCA GCA AGA CAG GCC AGA GTG TGG ACG AGC TCT 911

912 TCC AGA AAG TGG CAG AGG ATT ACG TCA GTG TGG CTG CCT TCC AGG TGA 959

960 TGA CAG AGG ACA AGG GCG TGG ATC TGG GCC AGA AGC CAA ACC CCT ACT 1007

1008 TCT ACA GCT GTT GTC ATC ACT GAG TCA GCA CTC ACC TGG CCT GGG GGA 1055

1056 ATT AAA GGA ATT CCC CGT AAG GGC TGG ACC CAG CTC CTT TCT GGG CTT 1103

1104 GGG TAG TCA AAT GTC TGA GCT ACC CCA GGT CCT CAT GTC AGC AGA GTG 1151

1152 GCG CCT GCC TGT GCT GGC CCA TGG AAC GGA GAC AGC ATT GGG CTG ACT 1199

1200 GTG GGC ATG AGG AGG GAT AAG GCT GAT TTG GAC CCC AGG CTT CTG CCC 1247

1248 TGG ACA GCA CTT GTG TCT GCA GAT TAT TTA AGT GGC TTT TGA TCT GTA 1295

1296 AAT AAA ATC AGT GCA CTG TGC ATC AAA AAA AAA AAA AAA AAA 1337

D.Blastp result

Query=PP4748 albumen (184 amino acid)

>SP_IN：P91857 P91857 caenorhabditis elegans.f26h9.6 protein.11/1999

Length=208 amino acid

Score value=108bits (266), predicated value=3e-23

Homogeny=60/140 (42%), similarity=88/140 (62%), breach=8/140 (5%)

Query：9 KVVMLGKEYVGKTSLVERYVHDRFLVGPYQ-NTIGAAFVAKVMSVGDRTVTLGIWDTAGS 67

K+V+LG+ VGK+SLV R+V +F YQ +TIGAAF+ + + + D T+ IWDTAG

Sbjct：21 KLVLLGESAVGKSSLVLRFVKGQF--HEYQESTIGAAFLTQTVCLDDATIKFEIWDTAGQ 78

Query：68 ERYEAMSRIYYRGAKAAIVCYDLTDSSSFERAKFWVKEL-RSLEEGCQIYLCGTKSXXXX 126

ERY +++ +YYRGA+AAIV YD+T+ SF++AK WVKEL R +L G K+

Sbjct：79 ERYHSLAPMYYRGAQAAIVVYDITNQESFQKAKNWVKELQRQASPNIVMALAGNKA---- 134

Query：127 XXXXXXXVDFHDVQDYADSS 146

V++ + YA+ +

Sbjct：135 DYANKRTVEYEEANAYAEDN 154

7.PP4762 albumen

A: nucleotide sequence (SEQ ID NO:19) length: 2417bp

GTTTCTGGCG CGAACTTCCG CCGTTCCGAA GTTGCACGGT GAATTGGCGC TATGTCTGGG 60

GACAGCAGCG GCCGCGGGCC AGAGGGCCGG GGCCGGGGCC GCGACCCGCA TCGGGATCGC 120

ACCCGCTCCC GCTCCCGCTC GCGGTCCCCT TTGTCGCCCA GGTCCCGCCG CGGCTCTGCG 180

CGGGAGCGCA GAGAGGCCCC AGAGCGCCCG AGCCTGGAGG ACACAGAGCC GTCGGATTCC 240

GGGGACGAGA TGATGGACCC GGCCAGCTTG GAGGCGGAGG CCGACCAAGG CCTGTGCCGC 300

CAGATCCGCC ATCAGTACCG GGCGCTCATC AACTCCGTCC AACGTAAGGC GGCGCCTCCG 360

GGCGGCGCGG GCCCGGACGG GCCGCTGTCC CCGCCCTGCG CCGGGCGCGG GGGGAGGGCA 420

CCGGAGGCAG GTGCCGGGCG GAGGCCCGGC GCGGCGTTAG TAGAGACGCG CCGGGTGCCA 480

GCCCCTCGCC GCACCTTAAA TTGTTGACAA AGTGCTCCCA CTTTGTGGCT GGAGCCGTAC 540

GACCTTGGCG CGGGTCCCCG AGTGGCGGGC TGGAAAAGCT GGAGGCCAGA GCCGTTCGCG 600

GACTCGCCCG AGCCCAGCCA GCCAGTGGGG AGAGGCAGGA CGCGGACTCC GCGTGCTTTG 660

ATTGCGCCCG GCAGTCCTCG GCGTGGGCTC CCGGCCTCGC AGCGGCACGC TGAGCTGCAG 720

CGGCACGCGA GAGCTGTCAA ATGCACGTTC TCGGACCCCA CCCGGACCCG CGGAATTCGA 780

ATCTCTGGGT GGGTCCGCGC AACTGTGGAG TGGCCCTCCA GGTGATGGTG TGTATTGCTT 840

GCAAAGTTTG AGAACCACTC AGTTATGGAG CACTCAGAGG CTGCAGCCGG AGTCCAGCCC 900

TTTGGGTTAA GGTTAGAGGG AGCTAGGGTT TCCTTTTAAA ATTCATGCTG CATAAAGGGT 960

AGGGTGGCCT TTCTCCTTTT CCGGGTCATT TTGGTTAAAA AGATAAAGAT GTTCGAAGTA 1020

ATTACTCAGA GCGCAGGGGC CGGGGCTTGG CTTTACTGTT GCAGGTGATA ACCTTGTCAA 1080

CTGCTTATTA GTGAATGCTG AACAAATTTC CCAAAGTTTG TATTTTTAAA AATAGAAAAC 1140

CGTGAGACAT ACTGAATGCC GGTGACAAAT TAACAGAGGT CCTTGAAGAG GCTAACACTC 1200

TGTTTAATGA AGTGTCCCGA GCAAGAGAAG CAGTCCTGGA TGCCCACTTT CTTGTTTTGG 1260

CTTCAGATTT GGGCAAAGAG AAAGCAAAGC AGCTGCGCTC AGACCTGAGC TCCTTTGACA 1320

TGTTAAGATA TGTTGAAACT CTACTCACAC ATATGGGTGT AAATCCGCTA GAAGCTGAAG 1380

AACTCATCCG TGATGAAGAT AGTCCTGATT TTGAATTCAT AGTCTATGAC TCCTGGAAGA 1440

TAACAGGCAG AACAGCAGAA AACACCTTTA ATAAAACCCA TACATTCCAC TTTCTGTTGG 1500

GTTCAATATA CGGAGAGTGC CCTGTGCCAA AGCCACGAGT TGATCGTCCA AGAAAAGTTC 1560

CTGTGATACA AGAGGAGAGG GCAATGCCTG CCCAGTTAAG AAGAATGGAA GAATCTCATC 1620

AAGAAGCAAC AGAGAAAGAA GTAGAAAGAA TCTTGGGATT GTTGCAGACA TATTTTCGAG 1680

AAGATCGAGA CAGTCTCACT CTTTCGCCCA GACTGGAATG CAGTGGCACC ATCTCAACTC 1740

ACTGCAACCT CCACCTCCTG GATTCAAGCA ATTCTCCTGC CTCAGCCTCC TGAGTAGCTG 1800

GGATTACAGG CGCACGCGCC ACCACATCTG GCTAATTTTT TTTCTGTATT TTTAGTAGAG 1860

ACATTGTTTC GCCATGTTGG CCAAGCTAGT CTTGAACCCC TGACCTCAGG TGATCTGCCA 1920

GCATCGGCCT CCCAAAGTGC TGGGATTACA GGTCTGATAC CCCAATGTCC TTCTTTGACT 1980

TTGTGGTTGA TCCTCATTCT TTCCCCCGTA CAGTGGAAAA CATCTTTCAT GTTTCCTTCA 2040

TTATACGGGA TGGTTTTGCA AGAATAAGAC TTGACCAAGA CCGACTGCCA GTAATAGAGC 2100

CTGTTAGTAT TAATGAAGAA AATGAGGGAT TTGAACATAA CACACAAGTT AGAAATCAAG 2160

GAATTATAGC TTTGAGTTAC CGTGACTGGG AGGAGATTGT GAAGACCTTT GAGATTTCAG 2220

AGCCTGTGAT TACTCCAAGT CAGAGGCAGC AGAAGCCAAG TGCTTGATGC TAGCTGAAGG 2280

ACTCAAATGG ATAGTGAAGT CCAAAACGGA AAGCGGCATG TATTGTACAT ATTGTATGAT 2340

TCAACATTTT TAAAGGCAGA TTGTTTTTAG TAAAATGTAG CTTTTGATAG TTAAAAAAAA 2400

AAAAAAAAAA AAAAAAA 2417

B: aminoacid sequence (SEQ ID NO:20) length: 157 amino acid

1 MLRYVETLLT HMGVNPLEAE ELIRDEDSPD FEFIVYDSWK ITGRTAENTF NKTHTFHFLL

61 GSIYGECPVP KPRVDRPRKV PVIQEERAMP AQLRRMEESH QEATEKEVER ILGLLQTYFR

121 EDRDSLTLSP RLECSGTIST HCNLHLLDSS NSPASAS

C. Nucleotide and amino acid composite sequence (SEQ ID NO:21)

Clone number: PP4762

Start code: 1320 ATG stop coding: 1793 TGA

Protein molecular weight: 18060

1 GT TTC TGG CGC GAA CTT CCG CCG TTC CGA AGT TGC ACG GTG AAT TGG 47

48 CGC TAT GTC TGG GGA CAG CAG CGG CCG CGG GCC AGA GGG CCG GGG CCG 95

96 GGG CCG CGA CCC GCA TCG GGA TCG CAC CCG CTC CCG CTC CCG CTC GCG 143

144 GTC CCC TTT GTC GCC CAG GTC CCG CCG CGG CTC TGC GCG GGA GCG CAG 191

192 AGA GGC CCC AGA GCG CCC GAG CCT GGA GGA CAC AGA GCC GTC GGA TTC 239

240 CGG GGA CGA GAT GAT GGA CCC GGC CAG CTT GGA GGC GGA GGC CGA CCA 287

288 AGG CCT GTG CCG CCA GAT CCG CCA TCA GTA CCG GGC GCT CAT CAA CTC 335

336 CGT CCA ACG TAA GGC GGC GCC TCC GGG CGG CGC GGG CCC GGA CGG GCC 383

384 GCT GTC CCC GCC CTG CGC CGG GCG CGG GGG GAG GGC ACC GGA GGC AGG 431

432 TGC CGG GCG GAG GCC CGG CGC GGC GTT AGT AGA GAC GCG CCG GGT GCC 479

480 AGC CCC TCG CCG CAC CTT AAA TTG TTG ACA AAG TGC TCC CAC TTT GTG 527

528 GCT GGA GCC GTA CGA CCT TGG CGC GGG TCC CCG AGT GGC GGG CTG GAA 575

576 AAG CTG GAG GCC AGA GCC GTT CGC GGA CTC GCC CGA GCC CAG CCA GCC 623

624 AGT GGG GAG AGG CAG GAC GCG GAC TCC GCG TGC TTT GAT TGC GCC CGG 671

672 CAG TCC TCG GCG TGG GCT CCC GGC CTC GCA GCG GCA CGC TGA GCT GCA 719

720 GCG GCA CGC GAG AGC TGT CAA ATG CAC GTT CTC GGA CCC CAC CCG GAC 767

768 CCG CGG AAT TCG AAT CTC TGG GTG GGT CCG CGC AAC TGT GGA GTG GCC 815

816 CTC CAG GTG ATG GTG TGT ATT GCT TGC AAA GTT TGA GAA CCA CTC AGT 863

864 TAT GGA GCA CTC AGA GGC TGC AGC CGG AGT CCA GCC CTT TGG GTT AAG 911

912 GTT AGA GGG AGC TAG GGT TTC CTT TTA AAA TTC ATG CTG CAT AAA GGG 959

960 TAG GGT GGC CTT TCT CCT TTT CCG GGT CAT TTT GGT TAA AAA GAT AAA 1007

1008 GAT GTT CGA AGT AAT TAC TCA GAG CGC AGG GGC CGG GGC TTG GCT TTA 1055

1056 CTG TTG CAG GTG ATA ACC TTG TCA ACT GCT TAT TAG TGA ATG CTG AAC 1103

1104 AAA TTT CCC AAA GTT TGT ATT TTT AAA AAT AGA AAA CCG TGA GAC ATA 1151

1152 CTG AAT GCC GGT GAC AAA TTA ACA GAG GTC CTT GAA GAG GCT AAC ACT 1199

1200 CTG TTT AAT GAA GTG TCC CGA GCA AGA GAA GCA GTC CTG GAT GCC CAC 1247

1248 TTT CTT GTT TTG GCT TCA GAT TTG GGC AAA GAG AAA GCA AAG CAG CTG 1295

1296 CGC TCA GAC CTG AGC TCC TTT GAC ATG TTA AGA TAT GTT GAA ACT CTA 1343

1 Met Leu Arg Tyr Val Glu Thr Leu 8

1344 CTC ACA CAT ATG GGT GTA AAT CCG CTA GAA GCT GAA GAA CTC ATC CGT 1391

9 Leu Thr His Met Gly Val Asn Pro Leu Glu Ala Glu Glu Leu Ile Arg 24

1392 GAT GAA GAT AGT CCT GAT TTT GAA TTC ATA GTC TAT GAC TCC TGG AAG 1439

25 Asp Glu Asp Ser Pro Asp Phe Glu Phe Ile Val Tyr Asp Ser Trp Lys 40

1440 ATA ACA GGC AGA ACA GCA GAA AAC ACC TTT AAT AAA ACC CAT ACA TTC 1487

41 Ile Thr Gly Arg Thr Ala Glu Asn Thr Phe Asn Lys Thr His Thr Phe 56

1488 CAC TTT CTG TTG GGT TCA ATA TAC GGA GAG TGC CCT GTG CCA AAG CCA 1535

57 His Phe Leu Leu Gly Ser Ile Tyr Gly Glu Cys Pro Val Pro Lys Pro 72

1536 CGA GTT GAT CGT CCA AGA AAA GTT CCT GTG ATA CAA GAG GAG AGG GCA 1583

73 Arg Val Asp Arg Pro Arg Lys Val Pro Val Ile Gln Glu Glu Arg Ala 88

1584 ATG CCT GCC CAG TTA AGA AGA ATG GAA GAA TCT CAT CAA GAA GCA ACA 1631

89 Met Pro Ala Gln Leu Arg Arg Met Glu Glu Ser His Gln Glu Ala Thr 104

1632 GAG AAA GAA GTA GAA AGA ATC TTG GGA TTG TTG CAG ACA TAT TTT CGA 1679

105 Glu Lys Glu Val Glu Arg Ile Leu Gly Leu Leu Gln Thr Tyr Phe Arg 120

1680 GAA GAT CGA GAC AGT CTC ACT CTT TCG CCC AGA CTG GAA TGC AGT GGC 1727

121 Glu Asp Arg Asp Ser Leu Thr Leu Ser Pro Arg Leu Glu Cys Ser Gly 136

1728 ACC ATC TCA ACT CAC TGC AAC CTC CAC CTC CTG GAT TCA AGC AAT TCT 1775

137 Thr Ile Ser Thr His Cys Asn Leu His Leu Leu Asp Ser Ser Asn Ser 152

1776 CCT GCC TCA GCC TCC TGA GTA GCT GGG ATT ACA GGC GCA CGC GCC ACC 1823

153 Pro Ala Ser Ala Ser *** 158

1824 ACA TCT GGC TAA TTT TTT TTC TGT ATT TTT AGT AGA GAC ATT GTT TCG 1871

1872 CCA TGT TGG CCA AGC TAG TCT TGA ACC CCT GAC CTC AGG TGA TCT GCC 1919

1920 AGC ATC GGC CTC CCA AAG TGC TGG GAT TAC AGG TCT GAT ACC CCA ATG 1967

1968 TCC TTC TTT GAC TTT GTG GTT GAT CCT CAT TCT TTC CCC CGT ACA GTG 2015

2016 GAA AAC ATC TTT CAT GTT TCC TTC ATT ATA CGG GAT GGT TTT GCA AGA 2063

2064 ATA AGA CTT GAC CAA GAC CGA CTG CCA GTA ATA GAG CCT GTT AGT ATT 2111

2112 AAT GAA GAA AAT GAG GGA TTT GAA CAT AAC ACA CAA GTT AGA AAT CAA 2159

2160 GGA ATT ATA GCT TTG AGT TAC CGT GAC TGG GAG GAG ATT GTG AAG ACC 2207

2208 TTT GAG ATT TCA GAG CCT GTG ATT ACT CCA AGT CAG AGG CAG CAG AAG 2255

2256 CCA AGT GCT TGA TGC TAG CTG AAG GAC TCA AAT GGA TAG TGA AGT CCA 2303

2304 AAA CGG AAA GCG GCA TGT ATT GTA CAT ATT GTA TGA TTC AAC ATT TTT 2351

2352 AAA GGC AGA TTG TTT TTA GTA AAA TGT AGC TTT TGA TAG TTA AAA AAA 2399

2400 AAA AAA AAA AAA AAA AAA 2417

8.PP5242 albumen

A: nucleotide sequence (SEQ ID NO:22) length: 1685bp

GCACCGCGGA GGACAGGGGC AGCTGGCGGG CAGCGGGTGA GGGGGTGGCG GGGACGCGAG 60

TGGCGGCCGC GGGGCCCCGG ACAAGGGTCC GCAGAGCTGC AGCCTTCGAG GGCCAGCCCT 120

CTCCGAGTCC GGGGCTGGGT CCCACCAGTG ACAAGGCGGC AGCCCCGCGC ACACCAAAGA 180

GAAGGCGGCT GTGGCGGCAG CGGCAGCCCC AGCCATGCTG TGTTATGTGA CGAGGCCGGA 240

CGCGGTGCTG ATGGAGGTGG AGGTGGAGGC GAAAGCCAAC GGCGAGGACT GCCTCAACCA 300

GGTGTGCAGG CGACTGGGAA TCATAGAAGT TGACTATTTT GGACTGCAGT TTACGGGTAG 360

CAAAGGTGAA AGTTTATGGC TAAACCTGAG AAACCGGATC TCCCAGCAGA TGGATGGGCT 420

AGCCCCTTAC AGGCTTAAAC TTAGAGTCAA GTTCTTCGTG GAGCCTCATC TCATCTTACA 480

GGAGCAGACT AGGCATATCT TTTTCTTGCA CATCAAGGAG GCCCTCTTGG CAGGCCACCT 540

CTTGTGTTCC CCAGAGCAGG CAGTGGAACT CAGTGCCCTC CTGGCCCAGA CCAAGTTTGG 600

AGACTACAAC CAGAACACTG CCAAGTATAA CTATGAGGAG CTCTGTGCCA AGGAGCTCTC 660

CTCTGCCACC TTGAACAGCA TTGTTGCAAA ACATAAGGAG TTGGAGGGGA CCAGCCAGGC 720

TTCAGCTGAA TACCAAGTTT TGCAGATTGT GTCGGCAATG GAAAACTATG GCATAGAATG 780

GCATTCTGTG CGGGATAGCG AAGGGCAGAA ACTGCTCATT GGGGTTGGAC CTGAAGGAAT 840

CTCAATTTGT AAAGATGACT TTAGCCCAAT TAATAGGATA GCTTATCCTG TGGTGCAGAT 900

GGCCACCCAG TCAGGAAAGA ATGTATATTT GACGGTCACC AAGGAATCTG GGAACAGCAT 960

CGTGCTCTTG TTTAAAATGA TCAGCACCAG GGCGGCCAGC GGGCTCTACC GAGCGATAAC 1020

AGAGACGCAC GCATTCTACA GGTGTGACAC AGTGACCAGC GCCGTGATGA TGCAGTATAG 1080

CCGTGACTTG AAGGGCCACT TGGCATCTCT GTTTCTGAAT GAAAACATTA ACCTTGGCAA 1140

GAAATATGTC TTTGATATTA AAAGAACATC AAAGGAGGTG TATGACCATG CCAGGAGGGC 1200

TCTGTACAAT GCTGGCGTTG TGGACCTCGT TTCAAGAAGC AACCAGAGCC CTTCACACTC 1260

GCCTCTGAAG TCCTCAGAAA GCAGCATGAA CTGCAGCAGC TGCGAGGGCC TCAGCTGCCA 1320

GCAGACCCGG GTGCTGCAGG AGAAGCTACG CAAGCTGAAG GAAGCCATGC TGTGCATGGT 1380

GTGCTGCGAG GAGGAGATCA ACTCCACCTT CTGTCCCTGT GGCCACACTG TGTGCTGTGA 1440

GAGCTGCGCC GCCCAGCTAC AGTCATGTCC CGTCTGCAGG TCGCGTGTGG AGCATGTCCA 1500

GCACGTCTAT CTGCCAACGC ACACCAGTCT TCTCAATCTG ACTGTAATCT AATCTGTTGT 1560

GCTTTTGTTG GACTTGGCAT GTTTCCATGA ACTGCACTAT TATAAACTAT TAAAATGATA 1620

GATTGTGGAG AAAGTAATTA TTCCAACACC CATCTGCCAT GCGATGTTAA AAAAAAAAAA 1680

AAAAA 1685

B: aminoacid sequence (SEQ ID NO:23) length: 445 amino acid

1 MLCYVTRPDA VLMEVEVEAK ANGEDCLNQV CRRLGIIEVD YFGLQFTGSK GESLWLNLRN

61 RISQQMDGLA PYRLKLRVKF FVEPHLILQE QTRHIFFLHI KEALLAGHLL CSPEQAVELS

121 ALLAQTKFGD YNQNTAKYNY EELCAKELSS ATLNSIVAKH KELEGTSQAS AEYQVLQIVS

181 AMENYGIEWH SVRDSEGQKL LIGVGPEGIS ICKDDFSPIN RIAYPVVQMA TQSGKNVYLT

241 VTKESGNSIV LLFKMISTRA ASGLYRAITE THAFYRCDTV TSAVMMQYSR DLKGHLASLF

301 LNENINLGKK YVFDIKRTSK EVYDHARRAL YNAGVVDLVS RSNQSPSHSP LKSSESSMNC

361 SSCEGLSCQQ TRVLQEKLRK LKEAMLCMVC CEEEINSTFC PCGHTVCCES CAAQLQSCPV

421 CRSRVEHVQH VYLPTHTSLL NLTVI

C. Nucleotide and amino acid composite sequence (SEQ ID NO:24)

Clone number: PP5242

Start code: 215 ATG stop coding: 1552 TAA

Protein molecular weight: 49881

1 G CAC CGC GGA GGA CAG GGG CAG CTG GCG GGC AGC GGG TGA GGG GGT 46

47 GGC GGG GAC GCG AGT GGC GGC CGC GGG GCC CCG GAC AAG GGT CCG CAG 94

95 AGC TGC AGC CTT CGA GGG CCA GCC CTC TCC GAG TCC GGG GCT GGG TCC 142

143 CAC CAG TGA CAA GGC GGC AGC CCC GCG CAC ACC AAA GAG AAG GCG GCT 190

191 GTG GCG GCA GCG GCA GCC CCA GCC ATG CTG TGT TAT GTG ACG AGG CCG 238

1 Met Leu Cys Tyr Val Thr Arg Pro 8

239 GAC GCG GTG CTG ATG GAG GTG GAG GTG GAG GCG AAA GCC AAC GGC GAG 286

9 Asp Ala Val Leu Met Glu Val Glu Val Glu Ala Lys Ala Asn Gly Glu 24

287 GAC TGC CTC AAC CAG GTG TGC AGG CGA CTG GGA ATC ATA GAA GTT GAC 334

25 Asp Cys Leu Asn Gln Val Cys Arg Arg Leu Gly Ile Ile Glu Val Asp 40

335 TAT TTT GGA CTG CAG TTT ACG GGT AGC AAA GGT GAA AGT TTA TGG CTA 382

41 Tyr Phe Gly Leu Gln Phe Thr Gly Ser Lys Gly Glu Ser Leu Trp Leu 56

383 AAC CTG AGA AAC CGG ATC TCC CAG CAG ATG GAT GGG CTA GCC CCT TAC 430

57 Asn Leu Arg Asn Arg Ile Ser Gln Gln Met Asp Gly Leu Ala Pro Tyr 72

431 AGG CTT AAA CTT AGA GTC AAG TTC TTC GTG GAG CCT CAT CTC ATC TTA 478

73 Arg Leu Lys Leu Arg Val Lys Phe Phe Val Glu Pro His Leu Ile Leu 88

479 CAG GAG CAG ACT AGG CAT ATC TTT TTC TTG CAC ATC AAG GAG GCC CTC 526

89 Gln Glu Gln Thr Arg His Ile Phe Phe Leu His Ile Lys Glu Ala Leu 104

527 TTG GCA GGC CAC CTC TTG TGT TCC CCA GAG CAG GCA GTG GAA CTC AGT 574

105 Leu Ala Gly His Leu Leu Cys Ser Pro Glu Gln Ala Val Glu Leu Ser 120

575 GCC CTC CTG GCC CAG ACC AAG TTT GGA GAC TAC AAC CAG AAC ACT GCC 622

121 Ala Leu Leu Ala Gln Thr Lys Phe Gly Asp Tyr Asn Gln Asn Thr Ala 136

623 AAG TAT AAC TAT GAG GAG CTC TGT GCC AAG GAG CTC TCC TCT GCC ACC 670

137 Lys Tyr Asn Tyr Glu Glu Leu Cys Ala Lys Glu Leu Ser Ser Ala Thr 152

671 TTG AAC AGC ATT GTT GCA AAA CAT AAG GAG TTG GAG GGG ACC AGC CAG 718

153 Leu Asn Ser Ile Val Ala Lys His Lys Glu Leu Glu Gly Thr Ser Gln 168

719 GCT TCA GCT GAA TAC CAA GTT TTG CAG ATT GTG TCG GCA ATG GAA AAC 766

169 Ala Ser Ala Glu Tyr Gln Val Leu Gln Ile Val Ser Ala Met Glu Asn 184

767 TAT GGC ATA GAA TGG CAT TCT GTG CGG GAT AGC GAA GGG CAG AAA CTG 814

185 Tyr Gly Ile Glu Trp His Ser Val Arg Asp Ser Glu Gly Gln Lys Leu 200

815 CTC ATT GGG GTT GGA CCT GAA GGA ATC TCA ATT TGT AAA GAT GAC TTT 862

201 Leu Ile Gly Val Gly Pro Glu Gly Ile Ser Ile Cys Lys Asp Asp Phe 216

863 AGC CCA ATT AAT AGG ATA GCT TAT CCT GTG GTG CAG ATG GCC ACC CAG 910

217 Ser Pro Ile Asn Arg Ile Ala Tyr Pro Val Val Gln Met Ala Thr Gln 232

911 TCA GGA AAG AAT GTA TAT TTG ACG GTC ACC AAG GAA TCT GGG AAC AGC 958

233 Ser Gly Lys Asn Val Tyr Leu Thr Val Thr Lys Glu Ser Gly Asn Ser 248

959 ATC GTG CTC TTG TTT AAA ATG ATC AGC ACC AGG GCG GCC AGC GGG CTC 1006

249 Ile Val Leu Leu Phe Lys Met Ile Ser Thr Arg Ala Ala Ser Gly Leu 264

1007 TAC CGA GCG ATA ACA GAG ACG CAC GCA TTC TAC AGG TGT GAC ACA GTG 1054

265 Tyr Arg Ala Ile Thr Glu Thr His Ala Phe Tyr Arg Cys Asp Thr Val 280

1055 ACC AGC GCC GTG ATG ATG CAG TAT AGC CGT GAC TTG AAG GGC CAC TTG 1102

281 Thr Ser Ala Val Met Met Gln Tyr Ser Arg Asp Leu Lys Gly His Leu 296

1103 GCA TCT CTG TTT CTG AAT GAA AAC ATT AAC CTT GGC AAG AAA TAT GTC 1150

297 Ala Ser Leu Phe Leu Asn Glu Asn Ile Asn Leu Gly Lys Lys Tyr Val 312

1151 TTT GAT ATT AAA AGA ACA TCA AAG GAG GTG TAT GAC CAT GCC AGG AGG 1198

313 Phe Asp Ile Lys Arg Thr Ser Lys Glu Val Tyr Asp His Ala Arg Arg 328

1199 GCT CTG TAC AAT GCT GGC GTT GTG GAC CTC GTT TCA AGA AGC AAC CAG 1246

329 Ala Leu Tyr Asn Ala Gly Val Val Asp Leu Val Ser Arg Ser Asn Gln 344

1247 AGC CCT TCA CAC TCG CCT CTG AAG TCC TCA GAA AGC AGC ATG AAC TGC 1294

345 Ser Pro Ser His Ser Pro Leu Lys Ser Ser Glu Ser Ser Met Asn Cys 360

1295 AGC AGC TGC GAG GGC CTC AGC TGC CAG CAG ACC CGG GTG CTG CAG GAG 1342

361 Ser Ser Cys Glu Gly Leu Ser Cys Gln Gln Thr Arg Val Leu Gln Glu 376

1343 AAG CTA CGC AAG CTG AAG GAA GCC ATG CTG TGC ATG GTG TGC TGC GAG 1390

377 Lys Leu Arg Lys Leu Lys Glu Ala Met Leu Cys Met Val Cys Cys Glu 392

1391 GAG GAG ATC AAC TCC ACC TTC TGT CCC TGT GGC CAC ACT GTG TGC TGT 1438

393 Glu Glu Ile Asn Ser Thr Phe Cys Pro Cys Gly His Thr Val Cys Cys 408

1439 GAG AGC TGC GCC GCC CAG CTA CAG TCA TGT CCC GTC TGC AGG TCG CGT 1486

409 Glu Ser Cys Ala Ala Gln Leu Gln Ser Cys Pro Val Cys Arg Ser Arg 424

1487 GTG GAG CAT GTC CAG CAC GTC TAT CTG CCA ACG CAC ACC AGT CTT CTC 1534

425 Val Glu His Val Gln His Val Tyr Leu Pro Thr His Thr Ser Leu Leu 440

1535 AAT CTG ACT GTA ATC TAA TCT GTT GTG CTT TTG TTG GAC TTG GCA TGT 1582

441 Asn Leu Thr Val Ile *** 446

1583 TTC CAT GAA CTG CAC TAT TAT AAA CTA TTA AAA TGA TAG ATT GTG GAG 1630

1631 AAA GTA ATT ATT CCA ACA CCC ATC TGC CAT GCG ATG TTA AAA AAA AAA 1678

1679 AAA AAA A 1685

D.Blastp result

Query=PP5242 albumen (445 amino acid)

>SP_IN：Q24440 Q24440 drosophila melanogaster(fruit fly).(cdnal)

protein 4.1 homologue(coracle).11/1999

Length=1698 amino acid

Score value=125bits (312), predicated value=4e-28

Homogeny=82/284 (28%), similarity=138/284 (47%), breach=8/284 (2%)

Query：2 LCYVTRPDAVLMEVEVEAKANGEDCLNQVCRRLGIIEVDYFGLQFTGSKGESLWLNLRNR 61

L VT D L++V ++ KA G D +N +C L +IE DYFGL + WL+L

Sbjct：33 LARVTLLDGSLLDVSIDRKAIGRDVINSICAGLNLIEKDYFGLTYETPTDPRTWLDLEKP 92

Query：62 ISQQMDGLAPYRLKLRVKFF-VEPHLILQEQTRHIFFLHIKEALLAGHLLCSPEQAVELS 120

+S + + L VKF+ EP + ++ TR+ L ++ +L G L C+ L

Sbjct：93 VS-KFFRTDTWPLTFAVKFYPPEPSQLKEDITRYHLCLQVRNDILEGRLPCTFVTHALLG 151

Query：121 ALLAQTKFGDYNQN---TAKYNYEELCAKELSSATLNSIVAKHKELEGTSQASAEYQVLQ 177

+ L Q++ GDY+ T Y+ A ++ + ++ HK +G S A AE L+

Sbjct：152 SYLVQSEMGDYDAEEMPTRAYLKDFKIAPNQTAELEDKVMDLHKTHKGQSPAEAELHYLE 211

Query：178 IVSAMENYGIEWHSVRDSEGQKLLIGVGPEGISICKDDFSPINRIAYPVVQMATQSGKNV 237

+ YG++ H +DSEG +++GV G+ + +D INR A+P + + +

Sbjct：212 NAKKLAMYGVDLHPAKDSEGVDIMLGVCASGLLVYRDKLR-INRFAWPKILKISYKRHHF 270

Query：238 YLTVTKESGNS I--VLLFKMISTRAASGLYRAITETHAFYRCDT 279

Y+ + + FK+ + RAA L+++ E H F+R T

Sbjct：271 YIKIRPGEFEQYESTIGFKLANHRAAKKLWKSCVEHHTFFRLMT 314

9.PP5656 albumen

A: nucleotide sequence (SEQ ID NO:25) length: 1373bp

TTCCGCAATT TCTACCTGAC CTTCCTGGAG TACGATGGGA ACCTGCTGCG GAGAGAGCTC 60

TTTGTGTGCC CCAGCCAGCC CCCACCTGGT GCTGAGCAGT TGCAGCAGGC CCTGGCACAA 120

CTGGACGAGG AAGACCCCTG CTTTGAGTTC CGGCAGCAGC AGCTCACTGT GCACCGTGTG 180

CATGTCACTT TCCTGCCCCA TGAACCGCCA CCCCCCCGGC CTCACGATGT CACCCTTGTG 240

GCCCAGCTGT CCATGGACCG GCTGCAGATG TTGGAAGCCC TGTGCAGGCA CTGGCCTGGC 300

CCCATGAGCC TGGCCTTGTA CCTGACAGAC GCAGAAGCTC AGCAGTTCCT GATTTCGGTC 360

GAGGCCTCAC CAGTGCTTGC TGCCCGGCAG GACGTGGCCT ACCATGTGGT GTACCGTGAG 420

GGCCCCTATA CCCCGTCAAC CAGCTTCGCA ACGTGGCCTT GGCCAGGCCC TCACGCCTTA 480

CGTCTTCCTC AGTGACATTG ACTTCCTGCC TGCCTATTCT CTCTACGACT ACCTCAGGGC 540

CTCCATTGAG CAGCTGGGGC TGGGCAGCCG GCGCAAGGCA GCACTGGTGG TGCCGGCATT 600

CGAGACCCTG CGCTACCGCT TCAGCTTCCC CCATTCCAAG GTGGAGCTGT TGGCCTTGCT 660

GGATGCGGGC ACTCTCTACA CCTTCAGGTA CCACGAGTGG CCCCGAGGCC ACGCACCCAC 720

AGACTATGCC CGCTGGCGGG AGGCTCAGGC CCCGTACCGT GTGCAATGGG CGGCCAACTA 780

TGAACCCTAC GTGGTGGTGC CACGAGACTG TCCCCGCTAT GATCCTCGCT TTGTGGGCTT 840

CCGCTGGAAC AAAGTGGCCC ACATTGTGGA GCTGGATGCC CAGGAATATG AGCTCCTGGT 900

GCTGCCCGAG GCCTTCACCA TCCATCTGCC CCACGCTCCA AGCCTGGACA TCTCCGCTTC 960

CGCTCCAGCC CCACCTATCG TGACTGCCTC CAGGCCCTCA AGGACGAATT CCACCAGGAC 1020

TTGTCCCGCC ACCATGGGGC TGCTGCCCTC AAATACCTCC CAGCCCTGCA GCAGCCCCAG 1080

AGCCCTGCCC GAGGCTGAGG CTGGGCCGGC GCTGCCCCTC ATCTTAGCAT TGGGCAGACA 1140

CCAGGGCAAC CTGCCCTCCG CCATCCCTGC TATTTAAATT ATTTAAGGTC TCTGGGAAGG 1200

GCTGGGGCAG AGCATCTGTG GGGTGGGGTC TTCCCCTTGC TGCTATTGTA TGGCTGGGGA 1260

CTGGTCTCTC TCTGCCCCAG CCAGTTTGGG GCTGGTTCCC CCATCTTGAA TTGTTTATCC 1320

CTTTTTCATA ATTAAAGTTT TAAAACATAA AAAAAAAAAA AAAAAAAAAA AAA 1373

B: aminoacid sequence (SEQ ID NO:26) length: 257 amino acid

1 MWCTVRAPIP RQPASQRGLG QALTPYVFLS DIDFLPAYSL YDYLRASIEQ LGLGSRRKAA

61 LVVPAFETLR YRFSFPHSKV ELLALLDAGT LYTFRYHEWP RGHAPTDYAR WREAQAPYRV

121 QWAANYEPYV VVPRDCPRYD PRFVGFGWNK VAHIVELDAQ EYELLVLPEA FTIHLPHAPS

181 LDISASAPAP PIVTASRPSR TNSTRTCPAT MGLLPSNTSQ PCSSPRALPE AEAGPALPLI

241 LALGRHQGNL PSAIPAI

C. Nucleotide and amino acid composite sequence (SEQ ID NO:27)

Clone number: PP5656

Start code: 404 ATG stop coding: 1177 TAA

Protein molecular weight: 28557

1 T TCC GCA ATT TCT ACC TGA CCT TCC TGG AGT ACG ATG GGA ACC TGC 46

47 TGC GGA GAG AGC TCT TTG TGT GCC CCA GCC AGC CCC CAC CTG GTG CTG 94

95 AGC AGT TGC AGC AGG CCC TGG CAC AAC TGG ACG AGG AAG ACC CCT GCT 142

143 TTG AGT TCC GGC AGC AGC AGC TCA CTG TGC ACC GTG TGC ATG TCA CTT 190

191 TCC TGC CCC ATG AAC CGC CAC CCC CCC GGC CTC ACG ATG TCA CCC TTG 238

239 TGG CCC AGC TGT CCA TGG ACC GGC TGC AGA TGT TGG AAG CCC TGT GCA 286

287 GGC ACT GGC CTG GCC CCA TGA GCC TGG CCT TGT ACC TGA CAG ACG CAG 334

335 AAG CTC AGC AGT TCC TGA TTT CGG TCG AGG CCT CAC CAG TGC TTG CTG 382

383 CCC GGC AGG ACG TGG CCT ACC ATG TGG TGT ACC GTG AGG GCC CCT ATA 430

1 Met Trp Cys Thr Val Arg Ala Pro Ile 9

431 CCC CGT CAA CCA GCT TCG CAA CGT GGC CTT GGC CAG GCC GTC ACG CCT 478

10 Pro Arg Gln Pro Ala Ser Gln Arg Gly Leu Gly Gln Ala Leu Thr Pro 25

479 TAC GTC TTC CTC AGT GAC ATT GAC TTC CTG CCT GCC TAT TCT CTC TAC 526

26 Tyr Val Phe Leu Ser Asp Ile Asp Phe Leu Pro Ala Tyr Ser Leu Tyr 41

527 GAC TAC CTC AGG GCC TCC ATT GAG CAG CTG GGG CTG GGC AGC CGG CGC 574

42 Asp Tyr Leu Arg Ala Ser Ile Glu Gln Leu Gly Leu Gly Ser Arg Arg 57

575 AAG GCA GCA CTG GTG GTG CCG GCA TTC GAG ACC CTG CGC TAC CGC TTC 622

58 Lys Ala Ala Leu Val Val Pro Ala Phe Glu Thr Leu Arg Tyr Arg Phe 73

623 AGC TTC CCC CAT TCC AAG GTG GAG CTG TTG GCC TTG CTG GAT GCG GGC 670

74 Ser Phe Pro His Ser Lys Val Glu Leu Leu Ala Leu Leu Asp Ala Gly 89

671 ACT CTC TAC ACC TTC AGG TAC CAC GAG TGG CCC CGA GGC CAC GCA CCC 718

90 Thr Leu Tyr Thr Phe Arg Tyr His Glu Trp Pro Arg Gly His Ala Pro 105

719 ACA GAC TAT GCC CGC TGG CGG GAG GCT CAG GCC CCG TAC CGT GTG CAA 766

106 Thr Asp Tyr Ala Arg Trp Arg Glu Ala Gln Ala Pro Tyr Arg Val Gln 121

767 TGG GCG GCC AAC TAT GAA CCC TAC GTG GTG GTG CCA CGA GAC TGT CCC 814

122 Trp Ala Ala Asn Tyr Glu Pro Tyr Val Val Val Pro Arg Asp Cys Pro 137

815 CGC TAT GAT CCT CGC TTT GTG GGC TTC GGC TGG AAC AAA GTG GCC CAC 862

138 Arg Tyr Asp Pro Arg Phe Val Gly Phe Gly Trp Asn Lys Val Ala His 153

863 ATT GTG GAG CTG GAT GCC CAG GAA TAT GAG CTC CTG GTG CTG CCC GAG 910

154 Ile Val Glu Leu Asp Ala Gln Glu Tyr Glu Leu Leu Val Leu Pro Glu 169

911 GCC TTC ACC ATC CAT CTG CCC CAC GCT CCA AGC CTG GAC ATC TCC GCT 958

170 Ala Phe Thr Ile His Leu Pro His Ala Pro Ser Leu Asp Ile Ser AIa 185

959 TCC GCT CCA GCC CCA CCT ATC GTG ACT GCC TCC AGG CCC TCA AGG ACG 1006

186 Ser Ala Pro Ala Pro Pro Ile Val Thr Ala Ser Arg Pro Ser Arg Thr 201

1007 AAT TCC ACC AGG ACT TGT CCC GCC ACC ATG GGG CTG CTG CCC TCA AAT 1054

202 Asn Ser Thr Arg Thr Cys Pro Ala Thr Met Gly Leu Leu Pro Ser Asn 217

1055 ACC TCC CAG CCC TGC AGC AGC CCC AGA GCC CTG CCC GAG GCT GAG GCT 1102

218 Thr Ser Gln Pro Cys Ser Ser Pro Arg Ala Leu Pro Glu Ala Glu Ala 233

1103 GGG CCG GCG CTG CCC CTC ATC TTA GCA TTG GGC AGA CAC CAG GGC AAC 1150

234 Gly Pro Ala Leu Pro Leu Ile Leu Ala Leu Gly Arg His Gln Gly Asn 249

1151 CTG CCC TCC GCC ATC CCT GCT ATT TAA ATT ATT TAA GGT CTC TGG GAA 1198

250 Leu Pro Ser Ala Ile Pro Ala Ile *** 258

1199 GGG CTG GGG CAG AGC ATC TGT GGG GTG GGG TCT TCC CCT TGC TGC TAT 1246

1247 TGT ATG GCT GGG GAC TGG TCT CTC TCT GCC CCA GCC AGT TTG GGG CTG 1294

1295 GTT CCC CCA TCT TGA ATT GTT TAT CCC TTT TTC ATA ATT AAA GTT TTA 1342

1343 AAA CAT AAA AAA AAA AAA AAA AAA AAA AAA A 1373

D.Blastp result

Query=PP5656 albumen (257 amino acid)

>SP_IN：Q21389 Q21389 caenorhabditis elegans.k09c8.4 protein.11/1999

Length=622 amino acid

Score value=104bits (256), predicated value=8e-22

Homogeny=65/166 (39%), similarity=93/166 (55%), breach=17/166 (10%)

Query：26 YVFLSDIDFLPAYSLYDYLRASIEQLGLGSRRKAALVVPAFET----LRYRFSFPHSKVE 81

Y+ ++D+DF+ L DY I+Q G ++K LV+PA E LR S S+ +

Sbjct：427 YILMTDVDFVV---LGDY-GTIIDQTG-NLKQKEVLVIPALEMTYPQLRLNLSNFLSRKD 481

Query：82 LLA--LLDAGTLYTFRYHEWPRGHAPTDYARWREAQAPY----RVQWAANYEPYVVVPRD 135

L+ LL+ T+ TFR WP H PT+ ++W ++ Y V + NYEPY V+ ++

Sbjct：482 LVIEHLLNK-TIQTFRETIWPSSHVPTNISKWIKSNRTYMVAQNVNYEKNYEPYFVIKKE 540

Query：136 -CPRYDPRFVGFGWNKVAHIVELDAQEYELLVLPEAFTIHLPHAPS 180

CP YD RF GFGWNKV H+++L Y+ LV P +F IH H S

Sbjct：541 ECPFYDQRFGGFGWNKVTHVMQLKMMNYKFLVSPTSFMIHQNHNAS 586

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.

Claims (10)

1. isolating human polypeptides with cancer suppressing function, it is characterized in that, it contains the aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, and described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.

2. polypeptide as claimed in claim 1, it is characterized in that this polypeptid acid sequence is selected from down group: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQID NO:26.

3. isolating polynucleotide is characterized in that, it is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3, it is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:26.

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

Coding region sequence or the full length sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27.

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 is a kind of host cell that is selected from down group:

(a) host cell that transforms or transduce with the described carrier of claim 6;

(b) host cell that transforms or transduce with the described polynucleotide of claim 3.

8. the preparation method of the polypeptide of the people's protein-active with cancer suppressing function is characterized in that this method comprises:

(a) have under the proteic condition of people of cancer suppressing function suitable the expression, cultivate the described host cell of claim 7;

(b) isolate polypeptide from culture, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivation.

9. energy and the described human polypeptides specificity bonded antibody with cancer suppressing function of claim 1, wherein said human polypeptides forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.

10. 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.