CN1193040C - New human protein with the function of inhibiting tumor 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 IDNO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20; 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.

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 IDNO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20.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.

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: the polypeptide of (b) isolating the protein-active with cancer suppressing function from culture.

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 PP3895 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 PP3993 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 IDNO: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) measure the level of the proteic transcript with 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, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; 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.

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.The people's protein level that is detected in the test with cancer suppressing function, 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 LaboratoryPress, 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

PP3895, PP3993, PP4052, PP4068, PP4135, PP4189, PP2500 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 Qiagen96 orifice plate plasmid extraction test kit, 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)
							PP3895	3	9	0	33	34	38
PP3993	17	8	12	35	24	32
							PP4052	0	1	0	16	20	18
PP4068	0	1	1	27	30	23
							PP4135	14	12	10	27	30	23
PP4189	6	5	9	27	30	23
							PP2500	0	2	0	28	30	27

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).

Embodiment 2: obtain gene clone by PCR from placenta cDNA:

Get the human placenta 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.Carry out reverse transcription reaction with MMLV-RT-SuperscriptH (GIBCO BRL) ThermoScript II at 42 ℃, obtain placenta cDNA.Utilize the different primer of commentaries on classics (as shown in the table) of each gene, by 90 ℃ of 3 ' 1 circulations.94 ℃ 30 seconds, 60 ℃ 30 seconds 72 ℃ 1 minute, totally 35 circulations, pcr amplification is carried out in 72 ℃ of 10 ' 1 circulations, obtains to contain the amplified production of each protein gene of complete open reading frame sequence.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, obtains recombinant protein.

The gene specific primer sequence

Clone's title	Special primer 1 (5 ' → 3 ')	Special primer 2 (5 ' → 3 ')
			PP3895	GGTTTACTGACACCCCCACCCCA	GCGCCCGGCCTCTTTTTATCCTT
PP3993	ACTTGCATTTGCCCTGACACCCA	GTTCTGCTTGGCCGAGCTGTTGA
			PP4052	GTGTATGCTGCCCCCTTTCTGGG	ACAGGATGGTAGTGGCGATGGCA
PP4068	CTGGGCCCAAGGACAAAGCTCAC	ATCATGGGGCATGCACAGCATCT
			PP4135	TGCCCCTAACCACTGAGACAGCA	CAACTGCACATTTTGCTCATGTA
PP4189	TCAAGGTTGCTCTCCAGCTCAAGG	GTTATTAGGCCCACCACTAAGAG
			PP2500	CTTGCTGCTCTTCTCGTTCCCGA	GCAGGGTCCTGGAACTTCTTGGC

Embodiment 3:cDNA cloned sequence is analyzed

1.PP3895 albumen

A: nucleotide sequence (SEQ ID NO:1) length: 1972bp

1 GGTTTAGTGA CACCCCCACC CCACCCCATC TGCATATTTT TTCACCACCC

51 CTCCCTTCTG TATATGATGC TTCTGTAGCT CTGTAACGCC CCCTACATTT

101 ACCTTCCTTA TATCTCCCCC GTCTTCCTCT CCATAGATCT CCTCCCATTT

151 CCCCTTCCAT GGTCCCCATC TTCCTTCTGA AATGTCTACT CCTTCATGTT

201 CCTTTATGTA TGTCTTCCAA TCTTTCCTTC CATAGCTCTC ATCACCTTCA

251 TATATTTCTT CCATCTTTCT CCTCCCACCT GCCTCGCCCT CTGTATATAC

301 CCCCACTCTC CCCCTTTTAT ATCTTCTCCA TCTCCCCCCA TATCTTTCCT

351 CTATGTCCAC ATCTGTGTAT TCCCCCCAAC TTCCCCTCCA TATATCTTTT

401 TTTACTCCCC TTTTCCTCCC TGTATCCTCT GTGTTCCCCC CATCTTGCTC

451 TACATCATTC TTCCCAAGAT CTTTACGTCT CCCATCTTGA TCTCTCCATC

501 TCCACTTTCT CCTAACATTT TCATTTCCGT TCCTTAGTGT CTCTAGAGAG

551 ATCATTCTTG ATAGCCTCAG CTCTTTCTCT GTGTTTTTCA GGTTTGTATT

601 CTGCTCTGCT CTACCTCTCC TCCTTGCCCC TTTTCTCTCC CAGGATGTCT

651 CTCCTTTCCA AATCCTTTTT GTACCTGAAT ACCTTTTGCC CCACCCTGGG

701 CTCTCATTTC CATCTCAGAC CTTAGCCTGG GATCTAAAGG GCTGACAGTG

751 TCCCTTTCTT CATGCAGATG ACAGTCGTCT AGAGGAGCTC AAAGCCACTC

801 TGCCCAGCCC AGACAAGCTC CCTGGATTCA AGATGTACCC CATTGACTTT

851 GAGAAGGTAT GGGGTGGGGC TCAGGACAGG GAAGGAGGAT GGGCAAAGCA

901 TAGACAGGCT GGAGAAAACA GAAGTATCTG GAGCCAGCCC CGGGCCTTTG

951 TGGGGATCAG ATTGTGGGCC TGCCATATGG CTCTGAATGA GTAGGTGTTC

1001 CCAGCCATCC CTTTGTGATC TGGGAGAGTC CAGCAGGCAA TTGCAGTGGA

1051 GGATACACAT CTTCTTTATC TGATCCTCTC CCCACTGCCT TCACACCCTC

1101 CCCACTCATA ACAGGATGAT GACAGCAACT TTCATATGGA TTTCATCGTG

1151 GCTGCATCCA ACCTCCGGGC AGAAAACTAT GACATTCCTT CTGCAGACCG

1201 GCACAAGAGC AAGCTGATTG CAGGGAAGAT CATCCCAGCC ATTGCCACGA

1251 CCACAGCAGC CGTGGTTGGC CTTGTGTGTC TGGAGCTGTA CAAGGTTGTG

1301 CAGGGGCACC GACAGCTTGA CTCCTACAAG AATGGTTTCC TCAACTTGGC

1351 CCTGCCTTTC TTTGGTTTCT CTGAACCCCT TGCCGCACCA CGTCACCAGG

1401 TGGGGGCCTG CATCCGAAGC AGGGTTTGGG TGGGGTGTAT CTGTGTAGAT

1451 CTGGTTCTGA TTCACGTCAT ACCCTGTCAC CAGGGGAGGG TTTCTGTCTG

1501 TGTACCTACC CTTTTTGTGT ATCCTTTTTC ACTTATTCAT TAATCACATT

1551 ATTTGAGTAC GTGCGAAAAG ATGGGATATT TGAATTGTGC CCTGGGAGAT

1601 TATTAGTAAC TACACAATAA TGGCAGCCAA AATTTATTGG ACGCTTCCTA

1651 CACTTAAGTG CTTTGCTTGC TTCATTAATG AATTCACTCA AATATTTATT

1701 GAGCACCTTT TGTGGGCAGG GACTCTTCTA AGTTATGTTC CTCAAGTAGA

1751 TTATATAAAT AACCTATTAA ATGATTTTGG AATCAAAAAA GGATAAAAAG

1801 AGGCCGGGCG CGGTGGCTTA CGCCTGTAAT CCCAGCACTT TGGGAGGCCG

1851 AGGCACGTGG TTCACCTGAA GTCAGGAGTT TGAGACCAGC CTGGCCAACA

1901 TGATGAAACC CTGTCTCTAC TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1951 AAAAAAAAAA AAAAAAAAAA AA

B: aminoacid sequence (SEQ ID NO:2) length: 135 amino acid

1 MDFIVAASNL RAENYDIPSA DRHKSKLIAG KIIPAIATTT AAVVGLVCLE LYKVVQGHRQ

61 LDSYKNGFLN LALPFFGFSE PLAAPRHQVG ACIRSRVWVG CICVDLVLIH VIPCHQGRVS

121 VCVPTLFVYP FSLIH

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

Clone number: PP3895

Start code: 1136 ATG stop coding: 1543 TAA

Protein molecular weight: 14799

1 G GTT TAC TGA CAC CCC CAC CCC ACC CCA TCT GCA TAT TTT TTC ACC 46

47 ACC CCT CCC TTC TGT ATA TGA TGC TTC TGT AGC TCT GTA ACG CCC CCT 94

95 ACA TTT ACC TTC CTT ATA TCT CCC CCG TCT TCC TCT CCA TAG ATC TCC 142

143 TCC CAT TTC CCC TTC CAT GGT CCC CAT CTT CCT TCT GAA ATG TCT ACT 190

191 CCT TCA TGT TCC TTT ATG TAT GTC TTC CAA TCT TTC CTT CCA TAG CTC 238

239 TCA TCA CCT TCA TAT ATT TCT TCC ATC TTT CTC CTC CCA CCT GCC TCG 286

287 CCC TCT GTA TAT ACC CCC ACT CTC CCC CTT TTA TAT CTT CTC CAT CTC 334

335 CCC CCA TAT CTT TCC TCT ATG TCC ACA TCT GTG TAT TCC CCC CAA CTT 382

383 CCC CTC CAT ATA TCT TTT TTT ACT CCC CTT TTC CTC CCT GTA TCC TCT 430

431 GTG TTC CCC CCA TCT TGC TCT ACA TCA TTC TTC CCA AGA TCT TTA CGT 478

479 CTC CCA TCT TGA TCT CTC CAT CTC CAC TTT CTC CTA ACA TTT TCA TTT 526

527 CCG TTC CTT AGT GTC TCT AGA GAG ATC ATT CTT GAT AGC CTC AGC TCT 574

575 TTC TCT GTG TTT TTC AGG TTT GTA TTC TGC TCT GCT CTA CCT CTC CTC 622

623 CTT GCC CCT TTT CTC TCC CAG GAT GTC TCT CCT TTC CAA ATC CTT TTT 670

671 GTA CCT GAA TAC CTT TTG CCC CAC CCT GGG CTC TCA TTT CCA TCT CAG 718

719 ACC TTA GCC TGG GAT CTA AAG GGC TGA CAG TGT CCC TTT CTT CAT GCA 766

767 GAT GAC AGT CGT CTA GAG GAG CTC AAA GCC ACT CTG CCC AGC CCA GAC 814

815 AAG CTC CCT GGA TTC AAG ATG TAC CCC ATT GAC TTT GAG AAG GTA TGG 862

863 GGT GGG GCT CAG GAC AGG GAA GGA GGA TGG GCA AAG CAT AGA CAG GCT 910

911 GGA GAA AAC AGA AGT ATC TGG AGC CAG CCC CGG GCC TTT GTG GGG ATC 958

959 AGA TTG TGG GCC TGC CAT ATG GCT CTG AAT GAG TAG GTG TTC CCA GCC 1006

1007 ATC CCT TTG TGA TCT GGG AGA GTC CAG CAG GCA ATT GCA GTG GAG GAT 1054

1055 ACA CAT CTT CTT TAT CTG ATC CTC TCC CCA CTG CCT TCA CAC CCT CCC 1102

1103 CAC TCA TAA CAG GAT GAT GAC AGC AAC TTT CAT ATG GAT TTC ATC GTG 1150

1 Met Asp Phe Ile Val 5

1151 GCT GCA TCC AAC CTC CGG GCA GAA AAC TAT GAC ATT CCT TCT GCA GAC 1198

6 Ala Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Ser Ala Asp 21

1199 CGG CAC AAG AGC AAG CTG ATT GCA GGG AAG ATC ATC CCA GCC ATT GCC 1246

22 Arg His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala Ile Ala 37

1247 ACG ACC ACA GCA GCC GTG GTT GGC CTT GTG TGT CTG GAG CTG TAC AAG 1294

38 Thr Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu Glu Leu Tyr Lys 53

1295 GTT GTG CAG GGG CAC CGA CAG CTT GAC TCC TAC AAG AAT GGT TTC CTC 1342

54 Val Val Gln Gly His Arg Gln Leu Asp Ser Tyr Lys Asn Gly Phe Leu 69

1343 AAC TTG GCC CTG CCT TTC TTT GGT TTC TCT GAA CCC CTT GCC GCA CCA 1390

70 Asn Leu Ala Leu Pro Phe Phe Gly Phe Ser Glu Pro Leu Ala Ala Pro 85

1391 CGT CAC CAG GTG GGG GCC TGC ATC CGA AGC AGG GTT TGG GTG GGG TGT 1438

86 Arg His Gln Val Gly Ala Cys Ile Arg Ser Arg Val Trp Val Gly Cys 101

1439 ATC TGT GTA GAT CTG GTT CTG ATT CAC GTC ATA CCC TGT CAC CAG GGG 1486

102 Ile Cys Val Asp Leu Val Leu Ile His Val Ile Pro Cys His Gln Gly 117

1487 AGG GTT TCT GTC TGT GTA CCT ACC CTT TTT GTG TAT CCT TTT TCA CTT 1534

118 Arg Val Ser Val Cys Val Pro Thr Leu Phe Val Tyr Pro Phe Ser Leu 133

1535 ATT CAT TAA TCA CAT TAT TTG AGT ACG TGC GAA AAG ATG GGA TAT TTG 1582

134 Ile His *** 136

1583 AAT TGT GCC CTG GGA GAT TAT TAG TAA CTA CAC AAT AAT GGC AGC CAA 1630

1631 AAT TTA TTG GAC GCT TCC TAC ACT TAA GTG CTT TGC TTG CTT CAT TAA 1678

1679 TGA ATT CAC TCA AAT ATT TAT TGA GCA CCT TTT GTG GGC AGG GAC TCT 1726

1727 TCT AAG TTA TGT TCC TCA AGT AGA TTA TAT AAA TAA CCT ATT AAA TGA 1774

1775 TTT TGG AAT CAA AAA AGG ATA AAA AGA GGC CGG GCG CGG TGG CTT ACG 1822

1823 CCT GTA ATC CCA GCA CTT TGG GAG GCC GAG GCA CGT GGT TCA CCT GAA 1870

1871 GTC AGG AGT TTG AGA CCA GCC TGG CCA ACA TGA TGA AAC CCT GTC TCT 1918

1919 ACT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1966

1967 AAA AAA 1972

2.PP3993 albumen

A: nucleotide sequence (SEQ ID NO:4) length: 2064bp

1 TTCAGACAAA CCTCAGGTAA GATGGGACTG GGCTTCCCCG CCACTGCGCA

51 GCAGCTCCCG CCCTCCTGGG TGCTGTCCCA GGGGTGAAAG GAAGAGGTGG

101 GGAGGTACAG CTGGGAGTGT GGGGGATGGG GAAGGATGGG GAGGGAACGG

151 GCCCGTGGAC AACTTGCATT TGCCCTGACA CCCACCCTCC CTGCAGCTCT

201 ACACCCTCAC CGTGATCGGC CCAGGACCGC CAGATTGCCA GCCAGCCCAG

251 ATCTCTCGCC GTTACTCGGA CTTTGAGCGG CTGCACCGAA ACCTGCAGCG

301 GCAATTCCGG GGCCCAATGG CTGCCATCTC CTTCCCCCGT AAGCGGCTGC

351 GCCGGAATTT TACTGCAGAG ACCATTGCCC GCCGTAGCCG GGCCTTTGAG

401 CAGTTTTTGG GTCACCTGCA GGCAGTGCCT GAGCTGCGCC ATGCCCCGGA

451 CCTGCAGGAC TTCTTCGTGC TGCCGGAGCT GCGGCGGGCA CAGAGCCTCA

501 CCTGTACTGG CCTCTATCGT GAGGCTCTGG CACTCTGGGC CAATGCCTGG

551 CAGCTGCAAG CCCAGCTGGG CACCCCCTCT GGCCCAGACC GCCCCCTGCT

601 GACCCTGGCT GGGCTGGCCG TGTGCCACCA GGAGCTGGAA GACCCTGGAG

651 AGGCCCGGGC ATGCTGTGAG AAGGCCCTGC AGCTGCTTGG GGACAAGAGC

701 CTCCACCCTT TGCTGGCACC CTTTCTGGAG GCCCATGTCC GGCTCTCCTG

751 GCGCCTGGGC CTGGACAAAC GTCAATCAGA GGCTCGGCTC CAAGCCCTGC

801 AGGAGGCAGG CCTTACCCCC ACACCACCCC CCAGTCTCAA AGAATTGCTC

851 ATCAAGGAGG TGCTGGACTA ACCCTTGCCT AGATTTAAGG CCACTGTGAG

901 GAGAGGGGTT GCCCCAGAAG GCAGGGGAAG GACCTGATGA GAACAGAATA

951 GCTGGGAGGC TGCAGAGGGT GCTGGGAGCC CCTAGAAGTT CCAAAAGAGA

1001 ATGTGAAGCA GATCAAGGAA ACTTCTGTTG AGCTAGGCTC AGGGTGAGCT

1051 TTGGCTGGGG TTGCCCTTGT GTAGTACAGG GAAGTCTGAC ACAGCCTCTC

1101 CAGCCTATAA ACAGCCGGGG GGCTGTGGCA CAGGTTGGGG CAATGTTCCC

1151 TTGTTGGTGG GCCCCCAAGC TGGCAAGGCC TCTTGGCTGA AGGCCAGGGA

1201 CTCTGCCCCT GGAGTCCTGG AGTTAAGGGA TGAAGGCAAG GCTGCAGGTC

1251 TGGCCCAGGG GAATTAAAAG CCAGCCACTC CAGTGGTATC AGTCTCTTTA

1301 TTGGATGTGA GGGCCAAAAG GGACTGTAAC TCCTGTCTCA GGAATGGGGA

1351 TAGATGGGAG GTTCTTGAAG CCCCAGGCGA AGCTGGTACC TCTGGCTACA

1401 GCTTGCTCTC TGAGACCTGG GGCTTCACTC GGATCACGCC CTCCTGGGCA

1451 CAGGTCACAG CTAGGACTCC ATCCTGACGC CACAGCCGCC CATGGACCAG

1501 CCCCCGAGAG CCACCTGTGG GTGAGGTGAA GGGTGATGAT GGCCTGCTTC

1551 AGAACAGCCA AATACACTTT TTTTTTTTTT CCTGAAACAG AGTCCCACTA

1601 AGTTGCCAGG CTGGTCTCAA GCCGCCTGGG TTCAAGGGAT CCTCCCGCCT

1651 CAGCCTCCTG AGCAGCTGGG ATTACAGGCG CACATCACCA TGCCCAACCT

1701 CCAAGTGGAC TTCTTGCAAA GGGTCTGGCC CAGGGCAGGG CTGCCCCACA

1751 CAAGGGTGCA CTGAGTGTCG TGGCTGCTCC AAATGCCCCT TCATGAGCTT

1801 ATTATGGACC GTCATTGAGG GGTAACTCCT CCCACAGGAA CCCCAGTTGA

1851 CAGTTTAAAA GCACTTTTAC ACCTCTCCTC GCTTCCTCAA AAAGATCACA

1901 GAGGGAGGAG CTCTGAGAAC AGTCTCCTTC AACAGCTCGG CCAAGCAGAA

1951 CTGCTGTACC TCTGACCACT TGTGTTAGGA AAACTATCGG CTCCCTGTAT

2001 AATAAATCAA GCCAGGTCCT CCCCAAAAAA AAAAAAAAAA AAAAAAAAAA

2051 AAAAAAAAAA AAAA

B: aminoacid sequence (SEQ ID NO:5) length: 184 amino acid

1 MAAISFPRKR LRRNFTAETI ARRSRAFEQF LGHLQAVPEL RHAPDLQDFF VLPELRRAQS

61 LTCTGLYREA LALWANAWQL QAQLGTPSGP DRPLLTLAGL AVCHQELEDP GEARACCEKA

121 LQLLGDKSLH PLLAPFLEAH VRLSWRLGLD KRQSEARLQA LQEAGLTPTP PPSLKELLIK

181 EVLD

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

Clone number: PP3993

Start code: 317 ATG stop coding: 871 TAA

Protein molecular weight: 20611

1 T TCA GAC AAA CCT CAG GTA AGA TGG GAC TGG GCT TCC CCG CCA CTG 46

47 CGC AGC AGC TCC CGC CCT CCT GGG TGC TGT CCC AGG GGT GAA AGG AAG 94

95 AGG TGG GGA GGT ACA GCT GGG AGT GTG GGG GAT GGG GAA GGA TGG GGA 142

143 GGG AAC GGG CCC GTG GAC AAC TTG CAT TTG CCC TGA CAC CCA CCC TCC 190

191 CTG CAG CTC TAC ACC CTC ACC GTG ATC GGC CCA GGA CCG CCA GAT TGC 238

239 CAG CCA GCC CAG ATC TCT CGC CGT TAC TCG GAC TTT GAG CGG CTG CAC 286

287 CGA AAC CTG CAG CGG CAA TTC CGG GGC CCA ATG GCT GCC ATC TCC TTC 334

1 Met Ala Ala Ile Ser Phe 6

335 CCC CGT AAG CGG CTG CGC CGG AAT TTT ACT GCA GAG ACC ATT GCC CGC 382

7 Pro Arg Lys Arg Leu Arg Arg Asn Phe Thr Ala Glu Thr Ile Ala Arg 22

383 CGT AGC CGG GCC TTT GAG CAG TTT TTG GGT CAC CTG CAG GCA GTG CCT 430

23 Arg Ser Arg Ala Phe Glu Gln Phe Leu Gly His Leu Gln Ala Val Pro 38

431 GAG CTG CGC CAT GCC CCG GAC CTG CAG GAC TTC TTC GTG CTG CCG GAG 478

39 Glu Leu Arg His Ala Pro Asp Leu Gln Asp Phe Phe Val Leu Pro Glu 54

479 CTG CGG CGG GCA CAG AGC CTC ACC TGT ACT GGC CTC TAT CGT GAG GCT 526

55 Leu Arg Arg Ala Gln Ser Leu Thr Cys Thr Gly Leu Tyr Arg Glu Ala 70

527 CTG GCA CTC TGG GCC AAT GCC TGG CAG CTG CAA GCC CAG CTG GGC ACC 574

71 Leu Ala Leu Trp Ala Asn Ala Trp Gln Leu Gln Ala Gln Leu Gly Thr 86

575 CCC TCT GGC CCA GAC CGC CCC CTG CTG ACC CTG GCT GGG CTG GCC GTG 622

87 Pro Ser Gly Pro Asp Arg Pro Leu Leu Thr Leu Ala Gly Leu Ala Val 102

623 TGC CAC CAG GAG CTG GAA GAC CCT GGA GAG GCC CGG GCA TGC TGT GAG 670

103 Cys His Gln Glu Leu Glu Asp Pro Gly Glu Ala Arg Ala Cys Cys Glu 118

671 AAG GCC CTG CAG CTG CTT GGG GAC AAG AGC CTC CAC CCT TTG CTG GCA 718

119 Lys Ala Leu Gln Leu Leu Gly Asp Lys Ser Leu His Pro Leu Leu Ala 134

719 CCC TTT CTG GAG GCC CAT GTC CGG CTC TCC TGG CGC CTG GGC CTG GAC 766

135 Pro Phe Leu Glu Ala His Val Arg Leu Ser Trp Arg Leu Gly Leu Asp 150

767 AAA CGT CAA TCA GAG GCT CGG CTC CAA GCC CTG CAG GAG GCA GGC CTT 814

151 Lys Arg Gln Ser Glu Ala Arg Leu Gln Ala Leu Gln Glu Ala Gly Leu 166

815 ACC CCC ACA CCA CCC CCC AGT CTC AAA GAA TTG CTC ATC AAG GAG GTG 862

167 Thr Pro Thr Pro Pro Pro Ser Leu Lys Glu Leu Leu Ile Lys Glu Val 182

863 CTG GAC TAA CCC TTG CCT AGA TTT AAG GCC ACT GTG AGG AGA GGG GTT 910

183 Leu Asp *** 185

911 GCC CCA GAA GGC AGG GGA AGG ACC TGA TGA GAA CAG AAT AGC TGG GAG 958

959 GCT GCA GAG GGT GCT GGG AGC CCC TAG AAG TTC CAA AAG AGA ATG TGA 1006

1007 AGC AGA TCA AGG AAA CTT CTG TTG AGC TAG GCT CAG GGT GAG CTT TGG 1054

1055 CTG GGG TTG CCC TTG TGT AGT ACA GGG AAG TCT GAC ACA GCC TCT CCA 1102

1103 GCC TAT AAA CAG CCG GGG GGC TGT GGC ACA GGT TGG GGC AAT GTT CCC 1150

1151 TTG TTG GTG GGC CCC CAA GCT GGC AAG GCC TCT TGG CTG AAG GCC AGG 1198

1199 GAC TCT GCC CCT GGA GTC CTG GAG TTA AGG GAT GAA GGC AAG GCT GCA 1246

1247 GGT CTG GCC CAG GGG AAT TAA AAG CCA GCC ACT CCA GTG GTA TCA GTC 1294

1295 TCT TTA TTG GAT GTG AGG GCC AAA AGG GAC TGT AAC TCC TGT CTC AGG 1342

1343 AAT GGG GAT AGA TGG GAG GTT CTT GAA GCC CCA GGC GAA GCT GGT ACC 1390

1391 TCT GGC TAC AGC TTG CTC TCT GAG ACC TGG GGC TTC ACT CGG ATC ACG 1438

1439 CCC TCC TGG GCA CAG GTC ACA GCT AGG ACT CCA TCC TGA CGC CAC AGC 1486

1487 CGC CCA TGG ACC AGC CCC CGA GAG CCA CCT GTG GGT GAG GTG AAG GGT 1534

1535 GAT GAT GGC CTG CTT CAG AAC AGC CAA ATA CAC TTT TTT TTT TTT TCC 1582

1583 TGA AAC AGA GTC CCA CTA AGT TGC CAG GCT GGT CTC AAG CCG CCT GGG 1630

1631 TTC AAG GGA TCC TCC CGC CTC AGC CTC CTG AGC AGC TGG GAT TAC AGG 1678

1679 CGC ACA TCA CCA TGC CCA ACC TCC AAG TGG ACT TCT TGC AAA GGG TCT 1726

1727 GGC CCA GGG CAG GGC TGC CCC ACA CAA GGG TGC ACT GAG TGT CGT GGC 1774

1775 TGC TCC AAA TGC CCC TTC ATG AGC TTA TTA TGG ACC GTC ATT GAG GGG 1822

1823 TAA CTC CTC CCA CAG GAA CCC CAG TTG ACA GTT TAA AAG CAC TTT TAC 1870

1871 ACC TCT CCT CGC TTC CTC AAA AAG ATC ACA GAG GGA GGA GCT CTG AGA 1918

1919 ACA GTC TCC TTC AAC AGC TCG GCC AAG CAG AAC TGC TGT ACC TCT GAC 1966

1967 CAC TTG TGT TAG GAA AAC TAT CGG CTC CCT GTA TAA TAA ATC AAG CCA 2014

2015 GGT CCT CCC CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2062

2063 AA 2064

3.PP4052 albumen

A: nucleotide sequence (SEQ ID NO:7) length: 1794bp

1 CTAAGAGAGC TTGGAAAGGG ATAGAGAAGT CTGACCCAAA TTTGCGGAGC

51 GACTGAGTGT ATGCTGCCCC CTTTCTGGGC CTTGGCTTCT TCCTCAATCA

101 TCTAGGCACA GTCCTATGAC TGCCTGTTTT TGAGGATGTG GGAAGGGTCT

151 GCAAATACAG TGCTTTCCCA TTGACACACG CTGGTGAGGA TGCAGGCTCC

201 CTGGCACCAG CAGTGAGGGC TCAGATTGCA AGAGTAAAAA CTTCCATCAC

251 TGGGAAGAGA AGTCTGCAGG GGACTGGAGG TGATCTGAAG ATTCTGAAAT

301 AACTCTTCCT CTCTCTGCAG AGAAGGATGG TGCTTCATCC TGTTTAGGTA

351 AGGGTGATAG CCAAGTGTGT TCAGGGTGGC TGCACCAACC CACCCTGAGT

401 CCATGTGGCT CAAGACTGCT GCTCAGGTGG GGTCAGCTGA GTGGGTAGGA

451 AGTCGGGAGG CACTGCCTAG CAGGTTTCAA CTTTAGTCTG GAGGCTGCAT

501 CTGTCTCCTC TAAACACAGT GGTTCTCATT GGTTATACCC CAAAATCACA

551 CCAGGAAACT TTAAAACTAG TGACGACTGA GTCCCGTCTC CAGAAACTCT

601 GACTTAATTG TGGGGGTGTG GCCTGGACAT CAGGATTTGT AATAACTCCT

651 CAGGTGATTC TCATGTAGTC AAGGTTGAGA ACCACTGCTT TAACTTTCTC

701 CAAATCCTAA TTACTTCTAG TGTGGCCTGA GGACTGCACT TTGAATGGCA

751 AGGGCCAAAT AACCAAGTTT TTGTTTTTTT CCAGGAAGTG GAGAAGAATT

801 TGGGGTTGGG ACTAGACTGG GGGTGAGCTG GGGAGATGGA AGATGGAAGT

851 GGGGTCAGTG GGAGGCAATG ATGGTAGGTA TCTTGGAAGA AGGATGCTTA

901 ATTTTAACAC GGAAAGACTG GAAGAAGGGA AGATACAAAG AGGTGGTCTC

951 CAGAAGACAG TCAGACAAAT AACAGAGCCT TAGAAATAAA ACCTTTTGGG

1001 CTGGGTGGCC CAGGTTCTCA ATTGCTGCCT CTACAGGAAA GTCTTTGGGT

1051 TCGGAGGATT CCAGAAACAT GAAGGAGAAG TTGGAGGACA TGGAGAGTGT

1101 CCTCAAGGAC CTGACAGAGG AGAAGAGAAA AGATGTGCTA AACTCCCTCG

1151 CTAAGTGCCT CGGCAAGGAG GATATTCGGC AGGATCTAGA GCAAAGAGTA

1201 TCTGAGGTCC TGATTTCCGG GGAGCTACAC ATGGAGGACC CAGACAAGCC

1251 TCTCCTAAGC AGCCTTTTTA ATGCTGCTGG GGTCTTGGTA GAAGCGCGTG

1301 CAAAAGCCAT TCTGGACTTC CTGGATGCCC TGCTAGAGCT GTCTGAAGAG

1351 CAGCAGTTTG TGGCTGAGGC CCTGGAGAAG GGGACCCTTC CTCTGTTGAA

1401 GGACCAGGTG AAATCTGTCA TGGAGCAGAA CTGGGATGAG CTGGCCAGCA

1451 GTCCTCCTGA CATGGACTAT GACCCTGAGG CACGAATTCT CTGTGCGCTG

1501 TATGTTGTTG TCTCTATCCT GCTGGAGCTG GCTGAGGGGC CTACCTCTGT

1551 CTCTTCCTAA CTACAAAAGC CCTTTCTCCC CACAAGCCTC TGGGTTTTCC

1601 CTTTACCAGT CTGTCCTCAC TGCCATCGCC ACTACCATCC TGTCACCAGT

1651 GGGACCTCTT TAAAACAAGC AGCCAACCAT TCTTTGATGT ATCCCATTCG

1701 CTCCATGTTA ACATCCAAAA CCAGCCTGGA TTTCATACAT GGACTTCTGA

1751 TTAAAAGTGG CAGGTTGTGC ATGTTAAAAA AAAAAAAAAA AAAA

B: aminoacid sequence (SEQ ID NO:8) length: 163 amino acid

1 MKEKLEDMES VLKDLTEEKR KDVLNSLAKC LGKEDIRQDL EQRVSEVLIS GELHMEDPDK

61 PLLSSLFNAA GVLVEARAKA ILDFLDALLE LSEEQQFVAE ALEKGTLPLL KDQVKSVMEQ

121 NWDELASSPP DMDYDPEARI LCALYVVVSI LLELAEGPTS VSS

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

Clone number: PP4052

Start code: 1069 ATG stop coding: 1560 TAA

Protein molecular weight: 18158

1 CTA AGA GAG CTT GGA AAG GGA TAG AGA AGT CTG ACC CAA ATT TGC GGA 48

49 GCG ACT GAG TGT ATG CTG CCC CCT TTC TGG GCC TTG GCT TCT TCC TCA 96

97 ATC ATC TAG GCA CAG TCC TAT GAC TGC CTG TTT TTG AGG ATG TGG GAA 144

145 GGG TCT GCA AAT ACA GTG CTT TCC CAT TGA CAC ACG CTG GTG AGG ATG 192

193 CAG GCT CCC TGG CAC CAG CAG TGA GGG CTC AGA TTG CAA GAG TAA AAA 240

241 CTT CCA TCA CTG GGA AGA GAA GTC TGC AGG GGA CTG GAG GTG ATC TGA 288

289 AGA TTC TGA AAT AAC TCT TCC TCT CTC TGC AGA GAA GGA TGG TGC TTC 336

337 ATC CTG TTT AGG TAA GGG TGA TAG CCA AGT GTG TTC AGG GTG GCT GCA 384

385 CCA ACC CAC CCT GAG TCC ATG TGG CTC AAG ACT GCT GCT CAG GTG GGG 432

433 TCA GCT GAG TGG GTA GGA AGT CGG GAG GCA CTG CCT AGC AGG TTT CAA 480

481 CTT TAG TCT GGA GGC TGC ATC TGT CTC CTC TAA ACA CAG TGG TTC TCA 528

529 TTG GTT ATA CCC CAA AAT CAC ACC AGG AAA CTT TAA AAC TAG TGA CGA 576

577 CTG AGT CCC GTC TCC AGA AAC TCT GAC TTA ATT GTG GGG GTG TGG CCT 624

625 GGA CAT CAG GAT TTG TAA TAA CTC CTC AGG TGA TTC TCA TGT AGT CAA 672

673 GGT TGA GAA CCA CTG CTT TAA CTT TCT CCA AAT CCT AAT TAC TTC TAG 720

721 TGT GGC CTG AGG ACT GCA CTT TGA ATG GCA AGG GCC AAA TAA CCA AGT 768

769 TTT TGT TTT TTT CCA GGA AGT GGA GAA GAA TTT GGG GTT GGG ACT AGA 816

817 CTG GGG GTG AGC TGG GGA GAT GGA AGA TGG AAG TGG GGT CAG TGG GAG 864

865 GCA ATG ATG GTA GGT ATC TTG GAA GAA GGA TGC TTA ATT TTA ACA CGG 912

913 AAA GAC TGG AAG AAG GGA AGA TAC AAA GAG GTG GTC TCC AGA AGA CAG 960

961 TCA GAC AAA TAA CAG AGC CTT AGA AAT AAA ACC TTT TGG GCT GGG TGG 1008

1009 CCC AGG TTC TCA ATT GCT GCC TCT ACA GGA AAG TCT TTG GGT TCG GAG 1056

1057 GAT TCC AGA AAC ATG AAG GAG AAG TTG GAG GAC ATG GAG AGT GTC CTC 1104

1 Met Lys Glu Lys Leu Glu Asp Met Glu Ser Val Leu 12

1105 AAG GAC CTG ACA GAG GAG AAG AGA AAA GAT GTG CTA AAC TCC CTC GCT 1152

13 Lys Asp Leu Thr Glu Glu Lys Arg Lys Asp Val Leu Asn Ser Leu Ala 28

1153 AAG TGC CTC GGC AAG GAG GAT ATT CGG CAG GAT CTA GAG CAA AGA GTA 1200

29 Lys Cys Leu Gly Lys Glu Asp Ile Arg Gln Asp Leu Glu Gln Arg Val 44

1201 TCT GAG GTC CTG ATT TCC GGG GAG CTA CAC ATG GAG GAC CCA GAC AAG 1248

45 Ser Glu Val Leu Ile Ser Gly Glu Leu His Met Glu Asp Pro Asp Lys 60

1249 CCT CTC CTA AGC AGC CTT TTT AAT GCT GCT GGG GTC TTG GTA GAA GCG 1296

61 Pro Leu Leu Ser Ser Leu Phe Asn Ala Ala Gly Val Leu Val Glu Ala 76

1297 CGT GCA AAA GCC ATT CTG GAC TTC CTG GAT GCC CTG CTA GAG CTG TCT 1344

77 Arg Ala Lys Ala Ile Leu Asp Phe Leu Asp Ala Leu Leu Glu Leu Ser 92

1345 GAA GAG CAG CAG TTT GTG GCT GAG GCC CTG GAG AAG GGG ACC CTT CCT 1392

93 Glu Glu Gln Gln Phe Val Ala Glu Ala Leu Glu Lys Gly Thr Leu Pro 108

1393 CTG TTG AAG GAC CAG GTG AAA TCT GTC ATG GAG CAG AAC TGG GAT GAG 1440

109 Leu Leu Lys Asp Gln Val Lys Ser Val Met Glu Gln Asn Trp Asp Glu 124

1441 CTG GCC AGC AGT CCT CCT GAC ATG GAC TAT GAC CCT GAG GCA CGA ATT 1488

125 Leu Ala Ser Ser Pro Pro Asp Met Asp Tyr Asp Pro Glu Ala Arg Ile 140

1489 CTC TGT GCG CTG TAT GTT GTT GTC TCT ATC CTG CTG GAG CTG GCT GAG 1536

141 Leu Cys Ala Leu Tyr Val Val Val Ser Ile Leu Leu Glu Leu Ala Glu 156

1537 GGG CCT ACC TCT GTC TCT TCC TAA CTA CAA AAG CCC TTT CTC CCC ACA 1584

157 Gly Pro Thr Ser Val Ser Ser *** 164

1585 AGC CTC TGG GTT TTC CCT TTA CCA GTC TGT CCT CAC TGC CAT CGC CAC 1632

1633 TAC CAT CCT GTC ACC AGT GGG ACC TCT TTA AAA CAA GCA GCC AAC CAT 1680

1681 TCT TTG ATG TAT CCC ATT CGC TCC ATG TTA ACA TCC AAA ACC AGC CTG 1728

1729 GAT TTC ATA CAT GGA CTT CTG ATT AAA AGT GGC AGG TTG TGC ATG TTA 1776

1777 AAA AAA AAA AAA AAA AAA 1794

4.PP4068 albumen

A: nucleotide sequence (SEQ ID NO:10) length: 1949bp

1 GGAAGGCAAA GGTAGAGCAA CTGGATCTCT GGCTCTCCAC ATAGCTTCTG

51 ATCTCAGACC TTACTAAAAT GCTTTCTGGG CCCAAGGACA AAGCTCACAT

101 GAACAAATGA TTTTGAGTCA TGAATGAAAA ATCTTGCTCT TTCCATAGTA

151 AAGAAGAATT AAGAGATGGA CAGGGTGAAA GATTGTCTGC TGGATATTCT

201 CCATCATATG ACAAGGACAA GAGTGTTCTG GCTTTCAGAG GAATCCCTAT

251 CTCAGAGTTG AAGAACCATG GCATTCTCCA GGCTCTGACC ACAGAAGCTT

301 ATGAATGGGA GCCACGTGTT GTGAGTACAG AGGTGGTCAG AGCCCAAGAA

351 GAATGGGAAG CTGTGGACAC CATCCAGCCA GAGACAGGGA GCCAAGCTAG

401 CTCAGAGCAG CCTGGGCAGC TAATCTCCTT CGGTGAGGCC CTGCAGCACT

451 TCCAGACTGT GGACCTTTCC CCCTTCAAGA AAAGAATCCA GCCAACTATT

501 CGAAGGACTG GGCTCGCCGC CCTCCGACAC TACCTCTTCG GGCCTCCAAA

551 GCTCCACCAG CGCCTTCGGG AAGAAAGGGA CTTGGTCCTG ACCATTGCTC

601 AGTGAGCGAA TCCAGCCACA GACCTGAGAG GCGCAGGCTT CCTTGCCCTC

651 CTGCATCTGC TCTACCTGGT GATGGACTCA AAGACCTTGC CGATGGCGCA

701 GGAGATTTTC CGCCTGTCTC GTCACCACAT CCAGCAATTC CCTTTCTGTT

751 TGATGTCCGT GAACATCACC CACATTGCCA TCCAGGCCTT GAGAGAGGAG

801 TGTCTCTCCA GAGAGTGTAA TCGGCAGCAG AAGGTCATCC CCGTGGTGAA

851 CAGCTTCTAT GCCGCCACAT TCCTCCACCT CGCACATGTC TGGAGGACAC

901 AGCGGAAGAC CATCTCAGAC TCGGGCTTTG TCCTCAAAGA GTTGGAAGTA

951 TTGGCCAAGA AGAGCCCACG GCGGCTGCTC AAGACCCTGG AGCTGTACTT

1001 GGCCAGGGTG TCAAAGGGAC AGGCCTCCTT GTTGGGAGCA CAGAAGTGCT

1051 ATGGGCCAGA AGCCCCTCCC TTCAAGGATC TCACCTTCAC AGGTGAGAGT

1101 GACCTGCAGT CTCACTCATC CGAAGGCGTA TGGCTGATCT GACCTCCGAG

1151 ATGAATGGAG GCTTAAAGGC TGAGCTGCAG GGGCTTTCAG GGGGTCAGTG

1201 GAGCCATGTC AGGAGCCTGG CCAGGCCGCA CCCCTTGCTG TCTCAGCAGA

1251 TGGGATATAG GAAGCTCCTG GGCTTAGCTG TGGGAAGCCA AGTACCCTCA

1301 CCGGCATGGG ACATGAGGGG CAGCTAGACT TCACCCCCTT CCCGCAGACC

1351 TGCCTCCAGA GCAAGGAGAA TTCTGCCTTA ATCTGTTGGG CTCCAGTCTC

1401 CGGGTTGAAT TCCAGTGTAT CCCACTGGGA GTGAATGGAT CATGAGGTGG

1451 GATGGCCCCA TCTGGATGTT CTGCAGATCC CCACATGGGA GGAGATTCCC

1501 AAGTAGAGGC AGCCAGAATT CTGACTCCCT GGCAGCAGCA GGGCTTTCAG

1551 GCACCAGTGT CTGTGTTGTT AGAACTCAGA GGAAGAGGCA GGGGCAGCAG

1601 CCGACAGGGG GCAGCATGAC CCAAGCAAGG GGCCTGAGGC CTCAGTGGGG

1651 GTAGGGCAAG GAGGTACCTC ACAGGTGGGT GTGAGGCCCC CTCTGGAGTT

1701 TCTGGCCATT CACTTACCCA CTCTCTTCTC CCCCTGACCC CCGCTCCATT

1751 GTTTATGATG GAAAAACGGA CATTTGGCTA GGTGTCTCTC ACGGCTGCTC

1801 CATCCTAGCC CCCACAAGCT GGGGCTTCCT CCTGTAGATG CTGTGCATGC

1851 CCCATGATGA GTTTCTGGCC TAATTGAGGG AAGGAGGAAA TTCATACCAG

1901 CAGTTTTCAA ATAAAAGAAT TGTTCTAATT AAAAAAAAAA AAAAAAAAA

B: aminoacid sequence (SEQ ID NO:11) length: 161 amino acid

1 MNEKSCSFHS KEELRDGQGE RLSAGYSPSY DKDKSVLAFR GIPISELKNH GILQALTTEA

61 YEWEPRVVST EVVRAQEEWE AVDTIQPETG SQASSEQPGQ LISFGEALQH FQTVDLSPFK

121 KRIQPTIRRT GLAALRHYLF GPPKLHQRLR EERDLVLTIA Q

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

Clone number: PP4068

Start code: 120 ATG stop coding: 605 TGA

Protein molecular weight: 18261

1 GG AAG GCA AAG GTA GAG CAA CTG GAT CTC TGG CTC TCC ACA TAG CTT 47

48 CTG ATC TCA GAC CTT ACT AAA ATG CTT TCT GGG CCC AAG GAC AAA GCT 95

96 CAC ATG AAC AAA TGA TTT TGA GTC ATG AAT GAA AAA TCT TGC TCT TTC 143

1 Met Asn Glu Lys Ser Cys Ser Phe 8

144 CAT AGT AAA GAA GAA TTA AGA GAT GGA CAG GGT GAA AGA TTG TCT GCT 191

9 His Ser Lys Glu Glu Leu Arg Asp Gly Gln Gly Glu Arg Leu Ser Ala 24

192 GGA TAT TCT CCA TCA TAT GAC AAG GAC AAG AGT GTT CTG GCT TTC AGA 239

25 Gly Tyr Ser Pro Ser Tyr Asp Lys Asp Lys Ser Val Leu Ala Phe Arg 40

240 GGA ATC CCT ATC TCA GAG TTG AAG AAC CAT GGC ATT CTC CAG GCT CTG 287

41 Gly Ile Pro Ile Ser Glu Leu Lys Asn His Gly Ile Leu Gln Ala Leu 56

288 ACC ACA GAA GCT TAT GAA TGG GAG CCA CGT GTT GTG AGT ACA GAG GTG 335

57 Thr Thr Glu Ala Tyr Glu Trp Glu Pro Arg Val Val Ser Thr Glu Val 72

336 GTC AGA GCC CAA GAA GAA TGG GAA GCT GTG GAC ACC ATC CAG CCA GAG 383

73 Val Arg Ala Gln Glu Glu Trp Glu Ala Val Asp Thr Ile Gln Pro Glu 88

384 ACA GGG AGC CAA GCT AGC TCA GAG CAG CCT GGG CAG CTA ATC TCC TTC 431

89 Thr Gly Ser Gln Ala Ser Ser Glu Gln Pro Gly Gln Leu Ile Ser Phe 104

432 GGT GAG GCC CTG CAG CAC TTC CAG ACT GTG GAC CTT TCC CCC TTC AAG 479

105 Gly Glu Ala Leu Gln His Phe Gln Thr Val Asp Leu Ser Pro Phe Lys 120

480 AAA AGA ATC CAG CCA ACT ATT CGA AGG ACT GGG CTC GCC GCC CTC CGA 527

121 Lys Arg Ile Gln Pro Thr Ile Arg Arg Thr Gly Leu Ala Ala Leu Arg 136

528 CAC TAC CTC TTC GGG CCT CCA AAG CTC CAC CAG CGC CTT CGG GAA GAA 575

137 His Tyr Leu Phe Gly Pro Pro Lys Leu His Gln Arg Leu Arg Glu Glu 152

576 AGG GAC TTG GTC CTG ACC ATT GCT CAG TGA GCG AAT CCA GCC ACA GAC 623

153 Arg Asp Leu Val Leu Thr Ile Ala Gln *** 162

624 CTG AGA GGC GCA GGC TTC CTT GCC CTC CTG CAT CTG CTC TAC CTG GTG 671

672 ATG GAC TCA AAG ACC TTG CCG ATG GCG CAG GAG ATT TTC CGC CTG TCT 719

720 CGT CAC CAC ATC CAG CAA TTC CCT TTC TGT TTG ATG TCC GTG AAC ATC 767

768 ACC CAC ATT GCC ATC CAG GCC TTG AGA GAG GAG TGT CTC TCC AGA GAG 815

816 TGT AAT CGG CAG CAG AAG GTC ATC CCC GTG GTG AAC AGC TTC TAT GCC 863

864 GCC ACA TTC CTC CAC CTC GCA CAT GTC TGG AGG ACA CAG CGG AAG ACC 911

912 ATC TCA GAC TCG GGC TTT GTC CTC AAA GAG TTG GAA GTA TTG GCC AAG 959

960 AAG AGC CCA CGG CGG CTG CTC AAG ACC CTG GAG CTG TAC TTG GCC AGG 1007

1008 GTG TCA AAG GGA CAG GCC TCC TTG TTG GGA GCA CAG AAG TGC TAT GGG 1055

1056 CCA GAA GCC CCT CCC TTC AAG GAT CTC ACC TTC ACA GGT GAG AGT GAC 1103

1104 CTG CAG TCT CAC TCA TCC GAA GGC GTA TGG CTG ATC TGA CCT CCG AGA 1151

1152 TGA ATG GAG GCT TAA AGG CTG AGC TGC AGG GGC TTT CAG GGG GTC AGT 1199

1200 GGA GCC ATG TCA GGA GCC TGG CCA GGC CGC ACC CCT TGC TGT CTC AGC 1247

1248 AGA TGG GAT ATA GGA AGC TCC TGG GCT TAG CTG TGG GAA GCC AAG TAC 1295

1296 CCT CAC CGG CAT GGG ACA TGA GGG GCA GCT AGA CTT CAC CCC CTT CCC 1343

1344 GCA GAC CTG CCT CCA GAG CAA GGA GAA TTC TGC CTT AAT CTG TTG GGC 1391

1392 TCC AGT CTC CGG GTT GAA TTC CAG TGT ATC CCA CTG GGA GTG AAT GGA 1439

1440 TCA TGA GGT GGG ATG GCC CCA TCT GGA TGT TCT GCA GAT CCC CAC ATG 1487

1488 GGA GGA GAT TCC CAA GTA GAG GCA GCC AGA ATT CTG ACT CCC TGG CAG 1535

1536 CAG CAG GGC TTT CAG GCA CCA GTG TCT GTG TTG TTA GAA CTC AGA GGA 1583

1584 AGA GGC AGG GGC AGC AGC CGA CAG GGG GCA GCA TGA CCC AAG CAA GGG 1631

1632 GCC TGA GGC CTC AGT GGG GGT AGG GCA AGG AGG TAC CTC ACA GGT GGG 1679

1680 TGT GAG GCC CCC TCT GGA GTT TCT GGC CAT TCA CTT ACC CAC TCT CTT 1727

1728 CTC CCC CTG ACC CCC GCT CCA TTG TTT ATG ATG GAA AAA CGG ACA TTT 1775

1776 GGC TAG GTG TCT CTC ACG GCT GCT CCA TCC TAG CCC CCA CAA GCT GGG 1823

1824 GCT TCC TCC TGT AGA TGC TGT GCA TGC CCC ATG ATG AGT TTC TGG CCT 1871

1872 AAT TGA GGG AAG GAG GAA ATT CAT ACC AGC AGT TTT CAA ATA AAA GAA 1919

1920 TTG TTC TAA TTA AAA AAA AAA AAA AAA AAA 1949

5.PP4135 albumen

A: nucleotide sequence (SEQ ID NO:13) length: 1585bp

1 CATACTTGGT CTATCTTCTA CTTTGTCTTC TCTTAGGACC CAAGGTCTCT

51 TAGCACAAAC ACTGCCTCTT GAGTTCCCAG TGCATCTGTA TCAATCTGGA

101 GATTACGTCC TCATCAAAAG CTGGAAAGAA GAAAAACTCG AACCAACCTG

151 GGAGACCTTA TCTAGTGCCC CTAACCACTG AGACAGCAGT CTGGACCGTT

201 AAGAAAGGGT AGACCCATCA CACTCAGGTG AAAAAGGCAT GACCCCCTTT

251 GGAGGCATAG GTTGTCACTC CCGGGCCAAC ACCTTCCAAA CTAATATTCA

301 AAAAAACTTA ACCTGTCTAA TTTGCTTCCT CTTTCTTTCG TTAGCTACCC

351 AGGAACATTT TATTTTATCA ATGTAACCTG ATCATCGTTT CCTCAAACAA

401 TTACATTTGA TGCTTGTCTT GTTATGCCCT GTGGGGACCT ACAAACCCAA

451 AGGCAACTAG CCTCTTCAGG CAATAACATA CAGGACATAG GAATGGGCAA

501 AGACTTCATG ACTAAAACAC CAAAAGCAAC AGCAACAAAA GCCAAAATTG

551 ACAAATGGGA TCTAATTAAA CTAAAGAGAT TCTGCGCAGC AAAGGAAACT

601 ATCATCAGAG TGAACAGGCA ACCTACAGAA TGGGAGAAAA TTTTTGCAAT

651 CTATCCATTT GACAAAGGGC TAATATCCAG AATCTATAAA GAACTTAAAT

701 TTACAAGAAA AAAACAACCC CATCAAAAAG CGGGCGAAGG ATATGAACAG

751 ACACTTCTCC AAAGAAGACA TTTATGCAGC CAACAAACAT GAAAAATAGC

801 TGATCATCAC TGGTCATTAT AGAAATGCAA ATCAAAACCA CAGTAAGATA

851 CTAACTCATG CCAGTTAGAA TGGCGATCAT TAAAAAGTCA GGAAACAACA

901 GATGCTGGAG AGGATGTGCA GAAATAGGAA TGCTTTTTAT ACTGTTGGTG

951 AAAGTGTAAA TTAGTTCAAC CATTGTGGAA GACAGTGTGG CGATTCCTCA

1001 AGGATCTATA GAACCAGAAC TACCACATGA CCCAGCAATC CCATTACTGG

1051 GTATATACCC AAAGGATTAT ACATCATTCT GCTATAAAGA CACATGCACA

1101 CGTACGTTTA TTGCAGCACT ATTTACAATA GCAAAGACTT GGAATCAACC

1151 CAAATCCCCG TCAATGATAG ACTGGATAAA GACAATGTGG CACTTATACA

1201 CCATGGAATA CTATGCAGCC AAAAAAAGGA TGAATTCATG TCCTTTGCAG

1251 CGACATGGAT GAAGCTGAAA ACCATCATTC TCAGCAAACT AACACGAGAA

1301 CAGAAAACCA AACACTACAT GTTCTCACTC ATAAGTGGGA GTTGAACAAT

1351 GAGAACACAT GGACACACGG AGGGGAACAC CACACACCAG GGCCTGTCGG

1401 CGGGTGGGGA GGCTAGGGGA GGGATAGCAT TAGGAGAAAT ACCTAATGTA

1451 GATGACAGGT TGATGGGTCT GACAAACCAC CATGACACGT GTATACCTAT

1501 GTAATGCAAC TGCACATTTT GCTCATGTAC CCCAGAACTT AAACTATAAT

1551 TAAAAACACA TAATTTCAAA AAAAAAAAAA AAAAA

B: aminoacid sequence (SEQ ID NO:14) length: 122 amino acid

1 MPCGDLQTQR QLASSGNNIQ DIGMGKDFMT KTPKATATKA KIDKWDLIKL KRFCAAKETI

61 IRVNRQPTEW EKIFAIYPFD KGLISRIYKE LKFTRKKQPH QKAGEGYEQT LLQRRHLCSQ

121 QT

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

Clone number: PP424

Start code: 424 ATG stop coding: 792 TGA

Protein molecular weight: 14172

1 CAT ACT TGG TCT ATC TTC TAC TTT GTC TTC TCT TAG GAC CCA AGG TCT 48

49 CTT AGC ACA AAC ACT GCC TCT TGA GTT CCC AGT GCA TCT GTA TCA ATC 96

97 TGG AGA TTA CGT CCT CAT CAA AAG CTG GAA AGA AGA AAA ACT CGA ACC 144

145 AAC CTG GGA GAC CTT ATC TAG TGC CCC TAA CCA CTG AGA CAG CAG TCT 192

193 GGA CCG TTA AGA AAG GGT AGA CCC ATC ACA CTC AGG TGA AAA AGG CAT 240

241 GAC CCC CTT TGG AGG CAT AGG TTG TCA CTC CCG GGC CAA CAC CTT CCA 288

289 AAC TAA TAT TCA AAA AAA CTT AAC CTG TCT AAT TTG CTT CCT CTT TCT 336

337 TTC GTT AGC TAC CCA GGA ACA TTT TAT TTT ATC AAT GTA ACC TGA TCA 384

385 TCG TTT CCT CAA ACA ATT ACA TTT GAT GCT TGT CTT GTT ATG CCC TGT 432

1 Met Pro Cys 3

433 GGG GAC CTA CAA ACC CAA AGG CAA CTA GCC TCT TCA GGC AAT AAC ATA 480

4 Gly Asp Leu Gln Thr Gln Arg Gln Leu Ala Ser Ser Gly Asn Asn Ile 19

481 CAG GAC ATA GGA ATG GGC AAA GAC TTC ATG ACT AAA ACA CCA AAA GCA 528

20 Gln Asp Ile Gly Met Gly Lys Asp Phe Met Thr Lys Thr Pro Lys Ala 35

529 ACA GCA ACA AAA GCC AAA ATT GAC AAA TGG GAT CTA ATT AAA CTA AAG 576

36 Thr Ala Thr Lys Ala Lys Ile Asp Lys Trp Asp Leu Ile Lys Leu Lys 51

577 AGA TTC TGC GCA GCA AAG GAA ACT ATC ATC AGA GTG AAC AGG CAA CCT 624

52 Arg Phe Cys Ala Ala Lys Glu Thr Ile Ile Arg Val Asn Arg Gln Pro 67

625 ACA GAA TGG GAG AAA ATT TTT GCA ATC TAT CCA TTT GAC AAA GGG CTA 672

68 Thr Glu Trp Glu Lys Ile Phe Ala Ile Tyr Pro Phe Asp Lys Gly Leu 83

673 ATA TCC AGA ATC TAT AAA GAA CTT AAA TTT ACA AGA AAA AAA CAA CCC 720

84 Ile Ser Arg Ile Tyr Lys Glu Leu Lys Phe Thr Arg Lys Lys Gln Pro 99

721 CAT CAA AAA GCG GGC GAA GGA TAT GAA CAG ACA CTT CTC CAA AGA AGA 768

100 His Gln Lys Ala Gly Glu Gly Tyr Glu Gln Thr Leu Leu Gln Arg Arg 115

769 CAT TTA TGC AGC CAA CAA ACA TGA AAA ATA GCT GAT CAT CAC TGG TCA 816

116 His Leu Cys Ser Gln Gln Thr *** 123

817 TTA TAG AAA TGC AAA TCA AAA CCA CAG TAA GAT ACT AAC TCA TGC CAG 864

865 TTA GAA TGG CGA TCA TTA AAA AGT CAG GAA ACA ACA GAT GCT GGA GAG 912

913 GAT GTG CAG AAA TAG GAA TGC TTT TTA TAC TGT TGG TGA AAG TGT AAA 960

961 TTA GTT CAA CCA TTG TGG AAG ACA GTG TGG CGA TTC CTC AAG GAT CTA 1008

1009 TAG AAC CAG AAC TAC CAC ATG ACC CAG CAA TCC CAT TAC TGG GTA TAT 1056

1057 ACC CAA AGG ATT ATA CAT CAT TCT GCT ATA AAG ACA CAT GCA CAC GTA 1104

1105 CGT TTA TTG CAG CAC TAT TTA CAA TAG CAA AGA CTT GGA ATC AAC CCA 1152

1153 AAT CCC CGT CAA TGA TAG ACT GGA TAA AGA CAA TGT GGC ACT TAT ACA 1200

1201 CCA TGG AAT ACT ATG CAG CCA AAA AAA GGA TGA ATT CAT GTC CTT TGC 1248

1249 AGC GAC ATG GAT GAA GCT GAA AAC CAT CAT TCT CAG CAA ACT AAC ACG 1296

1297 AGA ACA GAA AAC CAA ACA CTA CAT GTT CTC ACT CAT AAG TGG GAG TTG 1344

1345 AAC AAT GAG AAC ACA TGG ACA CAC GGA GGG GAA CAC CAC ACA CCA GGG 1392

1393 CCT GTC GGC GGG TGG GGA GGC TAG GGG AGG GAT AGC ATT AGG AGA AAT 1440

1441 ACC TAA TGT AGA TGA CAG GTT GAT GGG TCT GAC AAA CCA CCA TGA CAC 1488

1489 GTG TAT ACC TAT GTA ATG CAA CTG CAC ATT TTG CTC ATG TAC CCC AGA 1536

1537 ACT TAA ACT ATA ATT AAA AAC ACA TTT TTT CAA AAA AAA AAA AAA AAA 1584

1585 A 1585

6.PP4189 albumen

A: nucleotide sequence (SEQ ID NO:16) length: 1762bp

1 GAAAAAGGTA GAATTCTTAT GAATGTTTCA TGATCATGTA TGTGTGGTTA

51 ACGCAGATAC ATTTGGAAGT CGGTTATCAA GGTTGCTCTC CAGCTCAAGG

101 CCTTTGCCCT TTTTGTCAAA ACCAAAGAAG TTCCAACAAA AAGGAGTTTT

151 GAATGTAAAG AAAAATTGTG GAAATGCTGT CAGCAGCTAT TCACAGACCA

201 AACCAGCATC CATAGACATG TGGCAACACA ACATGCTGAT GAAATTTATC

251 ACCAGACAGC TTCTATTTTA AAGCAACTGG CTGTGACATT GAGCACCTCA

301 AAGAGTCTTT CGTCTGCAGA TGAAAAGAAC CCTTTAAAAG AGTGCCTTCC

351 ACATAGCCAT GACGTGTCTG CTTGGCTCCC TGATATAAGC TGCTTTAACC

401 CTGATGAGCT GATAAGTGGC CAGGGCAGTG AAGAAGGGGA GGTGCTCCTT

451 TATTACTGCT ACCATGACCT GGAGGATCCC CAATGGATCT GTGCCTGGCA

501 GACAGCTCTG TGTCAGCACC TGCACCTCAC AGGCAAGGAA TCCATTTATC

551 CCCAGGTGAA TTTCATAAAG AAGTAGAAAA GTTTTTATCT CAGGCAAATC

601 AAGAACAAAG TGATACTATC CTTCTTGATT GCAGAAACTT CTATGAAAGC

651 AAAATAGGAC GATTCCAAGG CTGCTTAGCC CCAGACATCA GGAAATTCAG

701 TTACTTCCCT AGCTACGTTG ACAAAAATCTAGAACTTTTC AGAGAGAAGA

751 GAGTGCTGAT GTACTGTACC GGGGGCATCC GCTGTGAGCG GGGTTCAGCC

801 TACCTCAAAG CCAAGGGAGT GTGCAAGGAG GTGTTCCAGC TCAAGGGTGG

851 CATCCACAAG TACCTGGAAG AGTTTCCTGA TGGCTTTTAC AAAGGGAAGT

901 TGTTTGTTTT TGATGAACGC TATGCTCTGT CCTACAACAG TGATGTGGTG

951 TCAGGTAGGT CAGCACAGGC TCAGAGCCCA AACTGAAATG AAGCACATTG

1001 TCAGTTCACC ATTCTAGAAA AATGACACAG GGAAGACAGG CCAGTGCTCA

1051 TTACTGAGCA CTGAATAAGC AGGGAAAATA AGTACATTGT GCCACCATTT

1101 TCCCAGCTGT GGAGCTGAGA GAACCCTAGC CCAGGAGTCA GGAGGCCTGG

1151 GTTGGGATCC TGGCTTCACC ATTGCTAGCT GGACAAGCCC ATTAACATGG

1201 GGATCATCTC ACCTGCCCTG CCTGCCTGTC TACCTGCCAA GAGCTGTACT

1251 ACTGGGCTAA TTCAGGGCTC TTAACCTGGA ATTGGTACAT AGATTTCAGG

1301 GATTCTGTGA ATTTGGATGG AAAAATAATT GTATCTTTGT TTTCAATAAC

1351 ACCTCACTAA AATGAAGCAT TTCCTTTAGT TATGAATGTA GGCAACAAAG

1401 TACCAGTTGT ATTAATGTAC CTGTGACTTT GTCTTCAGTA GGATTCACAA

1451 TACTTTCATA TCATGTTCTA GTTGCCTCAG ATATCTCAAA ATAGTATTTA

1501 TACTCATCAC TGCTTCAAAA TGAAAATAGT TATTAGGCCC ACCACTAAGA

1551 GTTGATATAT AATGTGTTAA TAAATGGCAC GTCTTATTAT ATATTACAGA

1601 TTTTGAAAAA GAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAACAAAAAA

1651 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1701 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1751 AAAAAAAAAA AA

B: aminoacid sequence (SEQ ID NO:17) length: 167 amino acid

1 MDLCLADSSV SAPAPHRQGI HLSPGEFHKE VEKFLSQANQ EQSDTILLDC RNFYESKIGR

61 FQGCLAPDIR KFSYFPSYVD KNLELFREKR VLMYCTGGIR CERGSAYLKA KGVCKEVFQL

121 KGGIHKYLEE FPDGFYKGKL FVFDERYALS YNSDVVSGRS AQAQSPN

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

Clone number: PP4198

Start code: 483 ATG stop coding: 986 TGA

Protein molecular weight: 19000

1 GA AAA AGG TAG AAT TCT TAT GAA TGT TTC ATG ATC ATG TAT GTG TGG 47

48 TTA ACG CAG ATA CAT TTG GAA GTC GGT TAT CAA GGT TGC TCT CCA GCT 95

96 CAA GGC CTT TGC CCT TTT TGT CAA AAC CAA AGA AGT TCC AAC AAA AAG 143

144 GAG TTT TGA ATG TAA AGA AAA ATT GTG GAA ATG CTG TCA GCA GCT ATT 191

192 CAC AGA CCA AAC CAG CAT CCA TAG ACA TGT GGC AAC ACA ACA TGC TGA 239

240 TGA AAT TTA TCA CCA GAC AGC TTC TAT TTT AAA GCA ACT GGC TGT GAC 287

288 ATT GAG CAC CTC AAA GAG TCT TTC GTC TGC AGA TGA AAA GAA CCC TTT 335

336 AAA AGA GTG CCT TCC ACA TAG CCA TGA CGT GTC TGC TTG GCT CCC TGA 383

384 TAT AAG CTG CTT TAA CCC TGA TGA GCT GAT AAG TGG CCA GGG CAG TGA 431

432 AGA AGG GGA GGT GCT CCT TTA TTA CTG CTA CCA TGA CCT GGA GGA TCC 479

480 CCA ATG GAT CTG TGC CTG GCA GAC AGC TCT GTG TCA GCA CCT GCA CCT 527

1 Met Asp Leu Cys Leu Ala Asp Ser Ser Val Ser Ala Pro Ala Pro 15

528 CAC AGG CAA GGA ATC CAT TTA TCC CCA GGT GAA TTT CAT AAA GAA GTA 575

16 His Arg Gln Gly Ile His Leu Ser Pro Gly Glu Phe His Lys Glu Val 31

576 GAA AAG TTT TTA TCT CAG GCA AAT CAA GAA CAA AGT GAT ACT ATC CTT 623

32 Glu Lys Phe Leu Ser Gln Ala Ash Gln Glu Gln Ser Asp Thr Ile Leu 47

624 CTT GAT TGC AGA AAC TTC TAT GAA AGC AAA ATA GGA CGA TTC CAA GGC 671

48 Leu Asp Cys Arg Asn Phe Tyr Glu Ser Lys Ile Gly Arg Phe Gln Gly 63

672 TGC TTA GCC CCA GAC ATC AGG AAA TTC AGT TAG TTC CCT AGC TAC GTT 719

64 Cys Leu Ala Pro Asp Ile Arg Lys Phe Ser Tyr Phe Pro Ser Tyr Val 79

720 GAC AAA AAT CTA GAA CTT TTC AGA GAG AAG AGA GTG CTG ATG TAC TGT 767

80 Asp Lys Asn Leu Glu Leu Phe Arg Glu Lys Arg Val Leu Met Tyr Cys 95

768 ACC GGG GGC ATC CGC TGT GAG CGG GGT TCA GCC TAC CTC AAA GCC AAG 815

96 Thr Gly Gly Ile Arg Cys Glu Arg Gly Ser Ala Tyr Leu Lys Ala Lys 111

816 GGA GTG TGC AAG GAG GTG TTC CAG CTC AAG GGT GGC ATC CAC AAG TAC 863

112 Gly Val Cys Lys Glu Val Phe Gln Leu Lys Gly Gly Ile His Lys Tyr 127

864 CTG GAA GAG TTT CCT GAT GGC TTT TAC AAA GGG AAG TTG TTT GTT TTT 911

128 Leu Glu Glu Phe Pro Asp Gly Phe Tyr Lys Gly Lys Leu Phe Val Phe 143

912 GAT GAA CGC TAT GCT CTG TCC TAC AAC AGT GAT GTG GTG TCA GGT AGG 959

144 Asp Glu Arg Tyr Ala Leu Ser Tyr Asn Ser Asp Val Val Ser Gly Arg 159

960 TCA GCA CAG GCT CAG AGC CCA AAC TGA AAT GAA GCA CAT TGT CAG TTC 1007

160 Ser Ala Gln Ala Gln Ser Pro Asn *** 168

1008 ACC ATT CTA GAA AAA TGA CAC AGG GAA GAC AGG CCA GTG CTC ATT ACT 1055

1056 GAG CAC TGA ATA AGC AGG GAA AAT AAG TAC ATT GTG CCA CCA TTT TCC 1103

1104 CAG CTG TGG AGC TGA GAG AAC CCT AGC CCA GGA GTC AGG AGG CCT GGG 1151

1152 TTG GGA TCC TGG CTT CAC CAT TGC TAG CTG GAC AAG CCC ATT AAC ATG 1199

1200 GGG ATC ATC TCA CCT GCC CTG CCT GCC TGT CTA CCT GCC AAG AGC TGT 1247

1248 ACT ACT GGG CTA ATT CAG GGC TCT TAA CCT GGA ATT GGT ACA TAG ATT 1295

1296 TCA GGG ATT CTG TGA ATT TGG ATG GAA AAA TAA TTG TAT CTT TGT TTT 1343

1344 CAA TAA CAC CTC ACT AAA ATG AAG CAT TTC CTT TAG TTA TGA ATG TAG 1391

1392 GCA ACA AAG TAC CAG TTG TAT TAA TGT ACC TGT GAC TTT GTC TTC AGT 1439

1440 AGG ATT CAC AAT ACT TTC ATA TCA TGT TCT AGT TGC CTC AGA TAT CTC 1487

1488 AAA ATA GTA TTT ATA CTC ATC ACT GCT TCA AAA TGA AAA TAG TTA TTA 1535

1536 GGC CCA CCA CTA AGA GTT GAT ATA TAA TGT GTT AAT AAA TGG CAC GTC 1583

1584 TTA TTA TAT ATT ACA GAT TTT GAA AAA GAA AAA AAA AAA AAA AAA AAA 1631

1632 AAA AAA AAA AAA CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1679

1680 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1727

1728 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA 1762

7.PP2500 albumen

A: nucleotide sequence (SEQ ID NO:19) length: 2153bp

1 CTGGGACGGG GGAAAGGAGA CGCTTCTTCC TCTTGCTGCT CTTCTCGTTC

51 CCGAGATCAG CGGCGGCGGT GACCGCGAGT GGGTCGGCAC CGTCTCCGGC

101 TCCGGGTGCG AACAATGCTG ACTGATAGCG GAGGCGGCGG CACCTCCTTT

151 GAGGAGGACC TGGACTCTGT GGCTCCGCGA TCCGCCCCAG CTGGGGCCTC

201 GGAGCCGCCT CCGCCGGGAG GGGTCGGTCT GGGGATCCGC ACCGTGAGGC

251 TCTTTGGGGA GGCCGGGCCA GCGTCGGGAG TCGGCAGCAG CGGCGGCGGC

301 GGCAGCGGCA GCGGTACGGG CGGAGGGGAC GCGGCGCTGG ATTTCAAGTT

351 GGCGGCTGCC GTGCTGAGGA CCGGGGGTGG AGGTGGTGCC TCTGGCAGTG

401 ACGAGGACGA AGTGTCCGAG GTTGAATCAT TTATTTTGGA CCAAGAAGAT

451 CTGGATAACC CAGTGCTTAA AACAACATCA GAGATATTCT TATCAAGTAC

501 TGCAGAAGGA GCAGACTTAC GCACTGTGGA TCCAGAGACA CAGGCACGAC

551 TAGAAGCATT GCTAGAAGCA GCAGGAATTG GCAAATTGTC AACTGCTGAT

601 GGTAAAGCTT TTGCAGATCC TGAGGTACTC CGGAGACTGA CATCCTCAGT

651 TAGTTGTGCA CTGGATGAAG CTGCTGCTGC ACTGACACGG ATGAAAGCAG

701 AAAACAGCCA CAATGCAGGA CAAGTGGACA CTCGCAGTCT AGCAGAAGCT

751 TGTTCAGATG GGGATGTTAA TGCTGTTCGT AAATTGCTAG ATGAAGGCAG

801 AAGTGTAAAT GAACATACAG AAGAAGGAGA AAGCCTGCTG TGTTTGGCTT

851 GTTCAGCAGG GTATTATGAA TTAGCACAAG TATTGCTTGC TATGCATGCT

901 AATGTTGAAG ATCGAGGGAA TAAAGGAGAC ATAACTCCCC TGATGGCAGC

951 TTCCAGTGGA GGTTACTTAG ATATTGTGAA ATTATTACTT CTTCATGATG

1001 CTGATGTCAA CTCCCAGTCT GCAACAGGAA ACACTGCGCT AACTTATGCA

1051 TGTGCTGGAG GATTTGTTGA CATTGTTAAA GTGCTCCTTA ATGAAGGTGC

1101 AAATATAGAA GATCATAATG AAAATGGACA TACTCCCTTA ATGGAAGCAG

1151 CCAGTGCAGG TCATGTGGAA GTTGCAAGAG TTCTTTTAGA TCATGGTGCA

1201 GGCATCAACA CTCATTCTAA TGAATTCAAA GAAAGTGCTC TAACACTTGC

1251 TTGCTACAAA GGCCATTTGG ATATGGTTCG CTTTCTACTT GAAGCTGGTG

1301 GAGATCAAGA GCACAAAACA GATGAGATGC ACACTGCCTT AATGGAGGCC

1351 TGCATGGATG GACATGTAGA GGTGGCACGT TTGCTTTTGG ATAGTGGTGC

1401 TCAAGTGAAC ATGCCTGCAG ATTCATTTGA ATCTCCATTG ACGCTAGCTG

1451 CCTGTGGAGG ACATGTTGAA TTGGCAGCTC TACTTATTGA AAGGGGAGCA

1501 AATCTTGAAG AAGTTAATGA TGAAGGATAC AGTCCCTTGA TGGAAGCTGC

1551 CCGGGAAGGA CATGAAGAAA TGGTGGCACT ACTCTTAGCA CAAGGAGCAA

1601 ATATAAATGC CCAGACAGAA GAAACTCAAG AAACTGCTCT TACCTTGGCT

1651 TGCTGTGGAG GATTTTCTGA AGTTGCAGAC TTTCTTATTA AGGCAGGGGC

1701 TGATATAGAA CTTGGCTGCT CCACACCTCT GATGGAGGCA TCTCAGGAGG

1751 GACACCTGGA ATTGGTTAAA TATTTGCTGG CTTCTGGCGC TAATGTGCAT

1801 GCTACAACAG CAACAGGAGA CACAGCCTTA ACCTATGCTT GTGAAAATGG

1851 ACATACGGAT GTTGCAGATG TTTTACTTCA AGCAGGGGCT GATTTAGACA

1901 AGCAGGAGGA CATGAAGACT ATTTTGGAGG GCATAGATCC GGCCAAGCAT

1951 CAGGTGAGGG TGGCCTTTGA TGCTTGTAAG CTACTACGTA AAGAATAGAT

2001 GTTGTAGGTA ACCAGAACTC TGGATATCTG AATTCCAGCC AAGAAGTTCC

2051 AGGACCCTGC TGGGTGACAA AGGAAATCCT CTTCAATTGA AAAAGATTAT

2101 GAAGTCCCAA TAAAAAGAGA TTTGTATTGC TAAAAAAAAA AAAAAAAAAA

2151 AAA

B: aminoacid sequence (SEQ ID NO:20) length: 627 amino acid

1 MLTDSGGGGT SFEEDLDSVA PRSAPAGASE PPPPGGVGLG IRTVRLFGEA GPASGVGSSG

61 GGGSGSGTGG GDAALDFKLA AAVLRTGGGG GASGSDEDEV SEVESFILDQ EDLDNPVLKT

121 TSEIFLSSTA EGADLRTVDP ETQARLEALL EAAGIGKLST ADGKAFADPE VLRRLTSSVS

181 CALDEAAAAL TRMKAENSHN AGQVDTRSLA EACSDGDVNA VRKLLDEGRS VNEHTEEGES

241 LLCLACSAGY YELAQVLLAM HANVEDRGNK GDITPLMAAS SGGYLDIVKL LLLHDADVNS

301 QSATGNTALT YACAGGFVDI VKVLLNEGAN IEDHNENGHT PLMEAASAGH VEVARVLLDH

361 GAGINTHSNE FKESALTLAC YKGHLDMVRF LLEAGADQEH KTDEMHTALM EACMDGHVEV

421 ARLLLDSGAQ VNMPADSFES PLTLAACGGH VELAALLIER GANLEEVNDE GYTPLMEAAR

481 EGHEEMVALL LAQGANINAQ TEETQETALT LACCGGFSEV ADFLIKAGAD IELGCSTPLM

541 EASQEGHLEL VKYLLASGAN VHATTATGDT ALTYACENGH TDVADVLLQA GADLDKQEDM

601 KTILEGIDPA KHQVRVAFDA CKLLRKE

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

Clone number: PP2500

Start code: 115 ATG stop coding: 1998 TAG

Protein molecular weight: 64908

1 CTG GGA CGG GGG AAA GGA GAC GCT TCT TCC TCT TGC TGC TCT TCT CGT 48

49 TCC CGA GAT CAG CGG CGG CGG TGA CCG CGA GTG GGT CGG CAC CGT CTC 96

97 CGG CTC CGG GTG CGA ACA ATG CTG ACT GAT AGC GGA GGC GGC GGC ACC 144

1 Met Leu Thr Asp Ser Gly Gly Gly Gly Thr 10

145 TCC TTT GAG GAG GAC CTG GAC TCT GTG GCT CCG CGA TCC GCC CCA GCT 192

11 Ser Phe Glu Glu Asp Leu Asp Ser Val Ala Pro Arg Ser Ala Pro Ala 26

193 GGG GCC TCG GAG CCG CCT CCG CCG GGA GGG GTC GGT CTG GGG ATC CGC 240

27 Gly Ala Ser Glu Pro Pro Pro Pro Gly Gly Val Gly Leu Gly Ile Arg 42

241 ACC GTG AGG CTC TTT GGG GAG GCC GGG CCA GCG TCG GGA GTC GGC AGC 288

43 Thr Val Arg Leu Phe Gly Glu Ala Gly Pro Ala Ser Gly Val Gly Ser 58

289 AGC GGC GGC GGC GGC AGC GGC AGC GGT ACG GGC GGA GGG GAC GCG GCG 336

59 Ser Gly Gly Gly Gly Ser Gly Ser Gly Thr Gly Gly Gly Asp Ala Ala 74

337 CTG GAT TTC AAG TTG GCG GCT GCC GTG CTG AGG ACC GGG GGT GGA GGT 384

75 Leu Asp Phe Lys Leu Ala Ala Ala Val Leu Arg Thr Gly Gly Gly Gly 90

385 GGT GCC TCT GGC AGT GAC GAG GAC GAA GTG TCC GAG GTT GAA TCA TTT 432

91 Gly Ala Ser Gly Ser Asp Glu Asp Glu Val Ser Glu Val Glu Ser Phe 106

433 ATT TTG GAC CAA GAA GAT CTG GAT AAC CCA GTG CTT AAA ACA ACA TCA 480

107 Ile Leu Asp Gln Glu Asp Leu Asp Asn Pro Val Leu Lys Thr Thr Ser 122

481 GAG ATA TTC TTA TCA AGT ACT GCA GAA GGA GCA GAC TTA CGC ACT GTG 528

123 Glu Ile Phe Leu Ser Ser Thr Ala Glu Gly Ala Asp Leu Arg Thr Val 138

529 GAT CCA GAG ACA CAG GCA CGA CTA GAA GCA TTG CTA GAA GCA GCA GGA 576

139 Asp Pro Glu Thr Gln Ala Arg Leu Glu Ala Leu Leu Glu Ala Ala Gly 154

577 ATT GGC AAA TTG TCA ACT GCT GAT GGT AAA GCT TTT GCA GAT CCT GAG 624

155 Ile Gly Lys Leu Ser Thr Ala Asp Gly Lys Ala Phe ALa Asp Pro Glu 170

625 GTA CTC CGG AGA CTG ACA TCC TCA GTT AGT TGT GCA CTG GAT GAA GCT 672

171 Val Leu Arg Arg Leu Thr Ser Ser Val Ser Cys Ala Leu Asp Glu Ala 186

573 GCT GCT GCA CTG ACA CGG ATG AAA GCA GAA AAC AGC CAC AAT GCA GGA 720

187 Ala Ala Ala Leu Thr Arg Met Lys Ala Glu Asn Ser His Asn Ala Gly 202

721 CAA GTG GAC ACT CGC AGT CTA GCA GAA GCT TGT TCA GAT GGG GAT GTT 768

203 Gln Val Asp Thr Arg Ser Leu Ala Glu Ala Cys Ser Asp Gly Asp Val 218

769 AAT GCT GTT CGT AAA TTG CTA GAT GAA GGC AGA AGT GTA AAT GAA CAT 816

219 Asn Ala Val Arg Lys Leu Leu Asp Glu Gly Arg Ser Val Asn Glu His 234

817 ACA GAA GAA GGA GAA AGC CTG CTG TGT TTG GCT TGT TCA GCA GGG TAT 864

235 Thr Glu Glu Gly Glu Ser Leu Leu Cys Leu Ala Cys Ser Ala Gly Tyr 250

865 TAT GAA TTA GCA CAA GTA TTG CTT GCT ATG CAT GCT AAT GTT GAA GAT 912

251 Tyr Glu Leu Ala Gln Val Leu Leu Ala Met His Ala Asn Val Glu Asp 266

913 CGA GGG AAT AAA GGA GAC ATA ACT CCC CTG ATG GCA GCT TCC AGT GGA 960

267 Arg Gly Asn Lys Gly Asp Ile Thr Pro Leu Met Ala Ala Ser Ser Gly 282

961 GGT TAC TTA GAT ATT GTG AAA TTA TTA CTT CTT CAT GAT GCT GAT GTC 1008

283 Gly Tyr Leu Asp Ile Val Lys Leu Leu Leu Leu His Asp Ala Asp Val 298

1009 AAC TCC CAG TCT GCA ACA GGA AAC ACT GCG CTA ACT TAT GCA TGT GCT 1056

299 Asn Ser Gln Ser Ala Thr Gly Asn Thr Ala Leu Thr Tyr Ala Cys Ala 314

1057 GGA GGA TTT GTT GAC ATT GTT AAA GTG CTC CTT AAT GAA GGT GCA AAT 1104

315 Gly Gly Phe Val Asp Ile Val Lys Val Leu Leu Asn Glu Gly Ala Asn 330

1105 ATA GAA GAT CAT AAT GAA AAT GGA CAT ACT CCC TTA ATG GAA GCA GCC 1152

331 Ile Glu Asp His Asn Glu Asn Gly His Thr Pro Leu Met Glu Ala Ala 346

1153 AGT GCA GGT CAT GTG GAA GTT GCA AGA GTT CTT TTA GAT CAT GGT GCA 1200

347 Ser Ala Gly His Val Glu Val Ala Arg Val Leu Leu Asp His Gly Ala 362

1201 GGC ATC AAC ACT CAT TCT AAT GAA TTC AAA GAA AGT GCT CTA ACA CTT 1248

363 Gly Ile Asn Thr His Ser Asn Glu Phe Lys Glu Ser Ala Leu Thr Leu 378

1249 GCT TGC TAC AAA GGC CAT TTG GAT ATG GTT CGC TTT CTA CTT GAA GCT 1296

379 Ala Cys Tyr Lys Gly His Leu Asp Met Val Arg Phe Leu Leu Glu Ala 394

1297 GGT GCA GAT CAA GAG CAC AAA ACA GAT GAG ATG CAC ACT GCC TTA ATG 1344

395 Gly Ala Asp Gln Glu His Lys Thr Asp Glu Met His Thr Ala Leu Met 410

1345 GAG GCC TGC ATG GAT GGA CAT GTA GAG GTG GCA CGT TTG CTT TTG GAT 1392

411 Glu Ala Cys Met Asp Gly His Val Glu Val Ala Arg Leu Leu Leu Asp 426

1393 AGT GGT GCT CAA GTG AAC ATG CCT GCA GAT TCA TTT GAA TCT CCA TTG 1440

427 Ser Gly Ala Gln Val Asn Met Pro Ala Asp Ser Phe Glu Ser Pro Leu 442

1441 ACG CTA GCT GCC TGT GGA GGA CAT GTT GAA TTG GCA GCT CTA CTT ATT 1488

443 Thr Leu Ala Ala Cys Gly Gly His Val Glu Leu Ala Ala Leu Leu Ile 458

1489 GAA AGG GGA GCA AAT CTT GAA GAA GTT AAT GAT GAA GGA TAC ACT CCC 1536

459 Glu Arg Gly Ala Asn Leu Glu Glu Val Asn Asp Glu Gly Tyr Thr Pro 474

1537 TTG ATG GAA GCT GCC CGG GAA GGA CAT GAA GAA ATG GTG GCA CTA CTC 1584

475 Leu Met Glu Ala Ala Arg Glu Gly His Glu Glu Met Val Ala Leu Leu 490

1585 TTA GCA CAA GGA GCA AAT ATA AAT GCC CAG ACA GAA GAA ACT CAA GAA 1632

491 Leu Ala Gln Gly Ala Asn Ile Asn Ala Gln Thr Glu Glu Thr Gln Glu 506

1633 ACT GCT CTT ACT TTG GCT TGC TGT GGA GGA TTT TCT GAA GTT GCA GAC 1680

507 Thr Ala Leu Thr Leu Ala Cys Cys Gly Gly Phe Ser Glu Val Ala Asp 522

1681 TTT CTT ATT AAG GCA GGG GCT GAT ATA GAA CTT GGC TGC TCC ACA CCT 1728

523 Phe Leu Ile Lys Ala Gly Ala Asp Ile Glu Leu Gly Cys Ser Thr Pro 538

1729 CTG ATG GAG GCA TCT CAG GAG GGA CAC CTG GAA TTG GTT AAA TAT TTG 1776

539 Leu Met Glu Ala Ser Gln Glu Gly His Leu Glu Leu Val Lys Tyr Leu 554

1777 CTG GCT TCT GGC GCT AAT GTG CAT GCT ACA ACA GCA ACA GGA GAC ACA 1824

555 Leu Ala Ser Gly Ala Asn Val His Ala Thr Thr Ala Thr Gly Asp Thr 570

1825 GCC TTA ACC TAT GCT TGT GAA AAT GGA CAT ACG GAT GTT GCA GAT GTT 1872

571 Ala Leu Thr Tyr Ala Cys Glu Asn Gly His Thr Asp Val Ala Asp Val 586

1873 TTA CTT CAA GCA GGG GCT GAT TTA GAC AAG CAG GAG GAC ATG AAG ACT 1920

587 Leu Leu Gln AAa Gly Ala Asp Leu Asp Lys Gln Glu Asp Met Lys Thr 602

1921 ATT TTG GAG GGC ATA GAT CCG GCC AAG CAT CAG GTG AGG GTG GCC TTT 1968

603 Ile Leu Glu Gly Ile Asp Pro Ala Lys His Gln Val Arg Val Ala Phe 618

1969 GAT GCT TGT AAG CTA CTA CGT AAA GAA TAG ATG TTG TAG GTA ACC AGA 2016

619 Asp Ala Cys Lys Leu Leu Arg Lys Glu *** 628

2017 ACT CTG GAT ATC TGA ATT CCA GCC AAG AAG TTC CAG GAC CCT GCT GGG 2064

2065 TGA CAA AGG AAA TCC TCT TCA ATT GAA AAA GAT TAT GAA GTC CCA ATA 2112

2113 AAA AGA GAT TTG TAT TGC TAA AAA AAA AAA AAA AAA AAA AA 2153

D.Blastp result

Query=PP2500 (627 amino acid)

>SP_IN：Q21920 Q21920 caenorhabditis elegans.r11a8.7 protein.11/1999

Length=2606 amino acid

Score value=184bits (462), predicated value=1e-45

Homogeny=115/306 (37%), similarity=168/306 (54%), breach=9/306 (2%)

Query：304 TGNTALTYACAGGFVDIVKVLLNEGANIEDHNENGHTPLMEAASAGHVEVARVLLDHGAG 363

T T LT ACA G DIV++LL EGANIE ++ G +PL+ AA+AGH V VLL + A

Sbjct：1220 TLETPLTIACANGHKDIVELLLKEGANIEHRDKKGFSPLIIAATAGHSSVVEVLLKNHAA 1279

Query：364 INTHSNEFKESALTLACYKGHLDMVRFLLEAGADQEHKTDEMHTALMEACMDGHVEVARL 423

I S+ K++AL+LAC G D+V LL GA++EH+ +T L A G++E+ +

Sbjct：1280 IEAQSDRTKDTALSLACSGGRKDVVELLLAHGANKEHRNVSDYTPLSLASSGGYIEIVNM 1339

Query：424 LLDSGAQVNMPADS--FESPLTLAACGGHVELAALLIERGANLE-EVNDEGYTPLMEAAR 480

LL +G+++N S SPL LA+ GH E +L+E+G+++ ++ T L A+

Sbjct：1340 LLTAGSEINSRTGSKLGISPLMLASMNGHREATRVLLEKGSDINAQIETNRNTALTLASF 1399

Query：481 EGHEEMVALLLAQGANINXXXXXXXXXXXXXXCCGGFSEVADFLIKAGAD-----IELGC 535

+G E+V LLLA AN+ GG+ +V +LI AGAD ++

Sbjct：1400 QGRTEVVKLLLAYNANVE-HRAKTGLTPLMECASGGYVDVGNLLIAAGADTNASPVQQTK 1458

Query：536 STPLMEASQEGHLELVKYLLASGANVHATTATGDTALTYACENGHTDVADVLLQAGADLD 595

T L ++++GH + V+ LL A V G TAL AC G+ A LL+GAD D

Sbjct：1459 DTALTISAEKGHEKFVRMLLNGDAAVDVRNKKGCTALWLACNGGYLSTAQALLEKGADPD 1518

Query：596 KQEDMK 601

++ K

Sbjct：1519 MFDNRK 1524

Score value=183bits (460), predicated value=2e-45

Homogeny=129/397 (32%), similarity=214/397 (53%), breach=18/397 (4%)

Query：170 EVLRRLTSSVSCALDEAAAALTRMKAENSHNAGQVDTRSLAEACSDGDVNAVRKLLDEGR 229

E+++ +S + ++ +L ++ GQ L S+GD +

Sbjct：1159 EIQKKGKTSSGTLISTSSKSLMAKSVQSQQQQGQ-----LRRTHSEGD--GAERAKSRSN 1211

Query：230 SVNEHTEEG-ESLLCLACSAGYYELAQVLLAMHANVEDRGNKGDITPLMAASSGGYLDIV 288

++++ TE E+ L +AC+ G+ ++ ++LL AN+E R KG +PL+ A++ G+ +V

Sbjct：1212 AIDKATETTLETPLTIACANGHKDIVELLLKEGANIEHRDKKG-FSPLIIAATAGHSSVV 1270

Query：289 KLLLLHDADVNSQS-ATGNTALTYACAGGFVDIVKVLLNEGANIEDHNENGHTPLMEAAS 347

++LL +A + +QS T +TAL+ AC+GG D+V++LL GAN E N+ +TPL A+S

Sbjct：1271 EVLLKNHAAIEAQSDRTKDTALSLACSGGRKDVVELLLAHGANKEHRNVSDYTPLSLASS 1330

Query：348 AGHVEVARVLLDHGAGINTHS-NEFKESALTLACYKGHLDMVRFLLEAGADQEHKTD-EM 405

G++E+ +LL G+ IN+ + ++ S L LA GH + R LLE G+D + +

Sbjct：1331 GGYIEIVNMLLTAGSEINSRTGSKLGISPLMLASMNGHREATRVLLEKGSDINAQIETNR 1390

Query：406 HTALMEACMDGHVEVARLLLDSGAQVNMPADSFESPLTLAACGGHVELAALLIERGA--N 463

+TAL A G EV+LLL A V A + +PL A GG+V++ LLI GA N

Sbjct：1391 NTALTLASFQGRTEVVKLLLAYNANVEHRAKTGLTPLMECASGGYVDVGNLLIAAGADTN 1450

Query：464 LEEVNDEGYTPLMEAAREGHEEMVALLLAQGANINXXXXXXXXXXXXXXCCGGFSEVADF 523

V T L +A +GHE+ V +LL A ++ C GG+ A

Sbjct：1451 ASPVQQTKDTALTISAEKGHEKFVRMLLNGDAAVD-VRNKKGCTALWLACNGGYLSTAQA 1509

Query：524 LIKAGADIELGCS---TPLMEASQEGHLELVKYLLAS 557

L++ GAD ++ + +P+M A ++GH+E+VKY++ S

Sbjct：1510 LLEKGADPDMFDNRKISPMMAAFRKGHVEIVKYMVNS 1546

Score value=160bits (400), predicated value=3e-38

Homogeny=120/400 (30%), similarity=206/400 (51%), breach=32/400 (8%)

Query：230 SVNEHTEEGESLLCLACSAGYYELAQVLLAMHANVEDRGNK-GDITPLMAASSGGYLDIV 288

S++ + ++L LA G + + + ++ RG+K ITPLM A++ IV

Sbjct：199 SIDSQIVQQNAMLLLAARVGIEQFVEYSHEIGV-MQFRGDKLSKITPLMEAAASSSETIV 257

Query：289 KLLLLHDADVNSQSATG-NTALTYACAGGFVDIVK-VLLNEGANIEDH---NENGHTPLM 343

+LL AD N S NTAL YA+ D+V+ +L +EG D N + H +M

Sbjct：258 RRLLELGADPNVASIPNCNTALIYAASTDGRDVVREILMTEGPKKPDVYLINNHYHDAMM 317

Query：344 EAASAGHVEVARVLLDHGAG---INTHSNEFKESALTLACYKGHLDMVRFLLEAGADQEH 400

E A G + + L+ G +N E ++SALTL+ KGH+ +V +++

Sbjct：318 EVALVGGTDTLKEFLEMGYRPRFLNLRQQE-RDSALTLSAQKGHIKIVTAIMDYYEKNPP 376

Query：401 KTDE--------MHTALMEACMDGHVEVARLLLDSGAQVNMPADSF--ESPLTLAACGGH 450

+T+E ++ALMEA M+GH++V +L+L G ++ + SPL +A+ GG+

Sbjct：377 QTEEEKQELCLERYSALMEAAMEGHIDVCKLMLSRGTPADLCTEVTIEPSPLIVASAGGY 436

Query：451 VELAALLIERGANLEEVNDEGYTPLMEAAREGHEE---MVALLLAQGANINXXXXXXXXX 507

E+ +L+ GA +EE++++ TPLMEA + +V LLL++ A ++

Sbjct：437 PEVVEVLLAAGAKIEELSNKKNTPLMEACAGDQGDQAGVVKLLLSKHAEVDVSNPDTGDT 496

Query：508 XXXXXCCGGFSEVADFLIKAGADIELGCSTPLMEASQEGHLELVKYLLASGANVHATTAT 567

G+ + LI+ G D+ G++P++EA++ GHLE ++++LA H T

Sbjct：497 PLSLAARNGYIAIMKMLIEKGGDLTAGKTSPIVEAARNGHLECIQFILA-----HCKTIP 551

Query：568 GD---TALTYACENGHTDVADVLLQAGADLDKQEDMKTIL 604

D AL A+G + + +++AGADL+ ++D +T L

Sbjct：552 QDQLSRALVSAADFGSLLIVEEVIRAGADLNFEQDERTAL 591

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, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivation.

2. polypeptide as claimed in claim 1 is characterized in that, this amino acid sequence of polypeptide is selected from down group: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:20.

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 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:17, SEQ IDNO:20.

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.

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 the polypeptide of the people's protein-active with cancer suppressing function from culture, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.

9. energy and the described human polypeptides specificity bonded antibody with cancer suppressing function of claim 1, wherein said polypeptide 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.