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

New human protein with the function of inhibiting cancer cell growth and its encoding sequence Download PDF

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CN1193041C
CN1193041C CNB001157450A CN00115745A CN1193041C CN 1193041 C CN1193041 C CN 1193041C CN B001157450 A CNB001157450 A CN B001157450A CN 00115745 A CN00115745 A CN 00115745A CN 1193041 C CN1193041 C CN 1193041C
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CN1324820A (en
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顾健人
杨胜利
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Shanghai Cancer Institute
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Shanghai Cancer Institute
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Abstract

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

Description

People's albumen and encoding sequence thereof with anticancer growth function
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to the proteic polynucleotide of people that new coding has cancer suppressing function, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.
Background technology
The research of people's gene group is international focus at present, removes human chromosome DNA large scale sequencing, outside the method for expressed sequence order-checking (EST), also lacks the screening that begins from function and has the high-throughout method of functional gene.
Cancer is one of principal disease of harm humans health.In order to treat effectively and prophylaxis of tumours, people more and more pay close attention to genetic treatment of tumor at present.Therefore, this area presses for people's albumen and the agonist/inhibitor thereof that development research has cancer suppressing function.
Summary of the invention
The purpose of this invention is to provide the new people's protein polypeptide of a class with cancer suppressing function with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated protein polypeptide with cancer suppressing function is provided, and it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26; Or its conservative property variation polypeptide or its active fragments or its reactive derivative.
Preferably, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:26.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned protein polypeptide with cancer suppressing function of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26.More preferably, the sequence of these polynucleotide is selected from down group: coding region sequence or the full length sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, the preparation method who prepares the polypeptide of the protein-active with cancer suppressing function is provided, this method comprises: (a) have under the proteic condition of cancer suppressing function suitable the expression, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate the polypeptide of protein-active with cancer suppressing function.
In a fifth aspect of the present invention, provide and above-mentioned protein polypeptide specificity bonded antibody with cancer suppressing function.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-800 Nucleotide in the above-mentioned polynucleotide.
In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the protein polypeptide and the pharmaceutically acceptable carrier with cancer suppressing function of the present invention of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer and cellular abnormality propagation.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Embodiment
The present invention adopts large-scale cDNA clone transfection cancer cells, has on the basis of cancer suppressing action in acquisition, proves new gene through order-checking, further obtains full length cDNA clone.DNA transfection evidence, the albumen with cancer suppressing function of the present invention has the effect that suppresses clone's formation to cancer cells (liver cancer cell), and its inhibiting rate is more than 50% or 50%.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating albumen or polypeptide with cancer suppressing function " is meant that the protein polypeptide with cancer suppressing function is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can have the albumen of cancer suppressing function with the purified technology of protein purifying of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.Purity with protein polypeptide of cancer suppressing function can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of the people with cancer suppressing function, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep natural identical biological function or the active polypeptide of people's albumen with cancer suppressing function of the present invention basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.With PP4277 albumen (in this application, its clone's numbering is adopted in proteinic name) (in this application, its clone numbering is adopted in proteinic name) be example, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.With PP4377 albumen (in this application, its clone's numbering is adopted in proteinic name) (in this application, its clone numbering is adopted in proteinic name) be example, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:6 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:5, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:6.Have the albumen of cancer suppressing function for other, can the rest may be inferred.Have the albumen of cancer suppressing function for other, can the rest may be inferred.
The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid has the proteic polynucleotide of cancer suppressing function to determine and/or to separate to encode.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
Dna sequence dna of the present invention can obtain with several method.For example, with hybridization technique DNA isolation well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homology nucleotide sequence and 2) antibody screening of expression library to be to detect the dna fragmentation of the clone with common structure feature.
The proteic specific DNA fragment sequence that coding has cancer suppressing function produces also and can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of required polypeptide.
In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.When if required amino acid whose whole sequence is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) mensuration has the level of the proteic transcript of cancer suppressing function; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.
In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 15 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, the length of probe within 2kb, preferably is within the 1kb usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.
In (4) kind method, detect the protein product of protein gene expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc. with cancer suppressing function.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The gene of the present invention that obtains as mentioned above, perhaps the available ordinary method of mensuration of the nucleotide sequence of various dna fragmentations etc. such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or albumen coded sequence with cancer suppressing function, and the method that produces polypeptide of the present invention through recombinant technology.
By the recombinant DNA technology of routine, can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the protein polypeptide with cancer suppressing function (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). have the proteic polynucleotide of people (or varient) of cancer suppressing function with coding of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the people's albumen polynucleotide sequence with cancer suppressing function can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people's encoding histone dna sequence dna with cancer suppressing function and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold SpringHarbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results 2Method is handled, and used step is well-known in this area.Alternative is to use MgCl 2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people's albumen or the polypeptide with cancer suppressing function of reorganization are of use in many ways.These purposes include, but is not limited to: directly have the disease due to the low or forfeiture of the protein function of cancer suppressing function as pharmacological agent and be used to screen and promote or antagonism has antibody, polypeptide or other part of the protein function of cancer suppressing function.For example, antibody can be used for activating or suppressing to have the proteic function of people of cancer suppressing function.The people's protein screening peptide library that has a cancer suppressing function with the reorganization of expressing can be used for seeking the peptide molecule that can suppress or stimulate the people's protein function with cancer suppressing function of therapeutic value.
The present invention also provides screening of medicaments to improve (agonist) or check the method that (antagonist) has the proteic medicament of people of cancer suppressing function to identify.Agonist improves the biological function such as stimulate cellular proliferation of the people's albumen with cancer suppressing function, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the proteic film preparation of people that mammalian cell or expression is had cancer suppressing function is cultivated with the people's albumen with cancer suppressing function of mark.Measure the medicine raising then or check this interactional ability.
The proteic antagonist of people with cancer suppressing function comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The proteic antagonist of people with cancer suppressing function can and be eliminated its function with the people's protein binding with cancer suppressing function, or suppresses to have the proteic generation of people of cancer suppressing function, or combines with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.The proteic antagonist of people with cancer suppressing function can be used for therepic use.
In screening during as the compound of antagonist, the albumen that can have a cancer suppressing function adds during bioanalysis measures, and determines by measuring albumen and the interaction between its acceptor that compounds affect has cancer suppressing function whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.
Polypeptide of the present invention can be directly used in disease treatment, for example, and various malignant tumours and cellular abnormality propagation etc.
Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.
Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound such as TNF etc.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.Albumen with cancer suppressing function comes administration with the amount that treats and/or prevents concrete indication effectively.The proteic amount with cancer suppressing function and the dosage range that are applied to the patient will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
The proteic polynucleotide of people with cancer suppressing function also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating since have that the proteic nothing of cancer suppressing function is expressed or the proteic expression with cancer suppressing function of unusual/non-activity due to cell proliferation, growth or metabolic disturbance.The albumen with cancer suppressing function that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic protein-active with cancer suppressing function.For example, a kind of albumen with cancer suppressing function of variation can be the albumen with cancer suppressing function that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the protein expression with cancer suppressing function or the disease of active caused by abnormal.Deriving from the expression vector of virus such as protein gene that retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for having cancer suppressing function is transferred in the cell.The method that structure carries the recombinant viral vector of the protein gene with cancer suppressing function is found in existing document (Sambrook, et al.).The people protein gene of reorganization with cancer suppressing function can be packaged in the liposome and be transferred in the cell in addition.
Suppress to have cancer suppressing function people's protein mRNA oligonucleotide (comprising sense-rna and DNA) and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carry out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension.
The present invention also provides the antibody at the people's proteantigen determinant with cancer suppressing function.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The anti-proteic antibody of people with cancer suppressing function can be used in the immunohistochemistry technology, detects the people's albumen with cancer suppressing function in the biopsy specimen.
With the also available labelled with radioisotope of the protein bound monoclonal antibody of the people with cancer suppressing function, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.
Antibody among the present invention can be used for treating or prevents and the relevant disease of people's albumen with cancer suppressing function.The antibody that gives suitable dosage can stimulate or block proteic generation of the people with cancer suppressing function or activity.
Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As have cancer suppressing function people's albumen high-affinity monoclonal antibody can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of the people's protein positive with cancer suppressing function.
Available people's albumen or the polypeptide immune animal of the production of polyclonal antibody with cancer suppressing function, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Have cancer suppressing function people's protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the anti-proteic single-chain antibody of people with cancer suppressing function.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of the people with cancer suppressing function obtains.During screening, must carry out mark to people's protein molecular with cancer suppressing function.
The invention still further relates to quantitatively and detection and localization has the diagnostic testing process of people's protein level of cancer suppressing function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.Detected in the test have cancer suppressing function-people's protein level, the disease that can have the importance of people's albumen in various diseases of cancer suppressing function with laying down a definition and be used to diagnose albumen to work with cancer suppressing function.
Proteic polynucleotide with cancer suppressing function can be used for having the diagnosis and the treatment of the protein related diseases of cancer suppressing function.Aspect diagnosis, the proteic polynucleotide with cancer suppressing function can be used for detecting have cancer suppressing function proteic expression whether or under morbid state, have an abnormal exprssion of cancer suppressing function.As the protein D NA sequence with cancer suppressing function can be used for the hybridization of biopsy specimen is had with judgement the proteic abnormal expression of cancer suppressing function.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of the albumen with cancer suppressing function and also can detect proteic transcription product with cancer suppressing function.
The sudden change that detection has the protein gene of cancer suppressing function also can be used for diagnosing the relevant disease of albumen with cancer suppressing function.Form with protein mutation of cancer suppressing function comprises that to have point mutation that the protein D NA sequence of cancer suppressing function compares, transposition, disappearance, reorganization and other any unusual etc. with normal wild type.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manual of BasicTechniques, Pergamon Press, New York (1988).
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Pyrenoids thuja acid full length sequence or its fragment with cancer suppressing function of the present invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
In addition, because the albumen with cancer suppressing function of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The acquisition of embodiment 1:cDNA gene and the restraining effect that the cancer cells clone is formed
PP4277, PP4377, PP4522, PP4583, PP4664, PP4748, PP4762, PP5242, PP5656 obtains by making up the human placenta cDNA library with ordinary method.Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.Make up the cDNA library of above-mentioned mRNA with pCMV-script TMXR cDNA library construction test kit (Stratagene company).Wherein ThermoScript II is used MMLV-RT-Superscript II (GIBCO BRL) instead, and reverse transcription reaction carries out at 42 ℃.Transform XL 10-Gold recipient cell, obtained 1 * 10 6The cDNA library of cfu/ μ g cDNA titre.The first round is picking cDNA clone at random, is probe with high abundance cDNA clone with the cDNA clone who has proved cancer inhibitor cell growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen 96 orifice plate plasmid extraction test kits, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously hepatoma cell line 7721.After the 100ng DNA alcohol precipitation drying, add 6 μ l H 2Transfection is treated in the O dissolving.Add 0.74 μ l liposome and 9.3 μ l serum-free mediums in every part of DNA sample, behind the mixing, room temperature was placed 10 minutes.Add 150 μ l serum-free mediums in every pipe, divide equally and add 3 holes and grow in 7721 cells of 96 orifice plates, placed 2 hours for 37 ℃, every hole adds 50 μ l serum-free mediums again, 37 ℃ 24 hours.Every hole is changed 100 μ l and is trained liquid entirely, 37 ℃ 24 hours, change the full training liquid 100 μ l that contain G418,37 ℃ 24 ~ 48 hours, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2 ~ 3 times, there is the clone to form up to the microscopy cell, counting.Find that above clone has the cell clone of inhibition formation effect, the result is as shown in the table.
CDNA clone's transfectional cell (7721) clone formation situation
CDNA clones title C DNA cloning number (three repetitions) Empty carrier clone number (three repetitions)
PP4277 0 0 4 45 37 30
PP4377 1 16 4 33 12 16
PP4522 0 0 0 23 21 14
PP4583 0 0 0 23 21 14
PP4664 7 6 1 32 34 29
PP4748 0 2 1 21 14 18
PP4762 4 11 0 32 29 43
PP5242 3 4 3 16 13 9
PP5656 0 0 0 35 39 32
The cDNA clone is adopted two deoxidation cessation method, on the ABI377 automatic dna sequencer, measure the nucleotide sequence of the nearly 500bp of one end.After the analysis, be defined as novel gene cloning, carry out the other end order-checking.As obtaining full length cDNA sequence not yet, then design primer, check order once more, up to obtaining full length sequence (SEQ ID NO:1,4,7,10,13,16,19,22,24).
Embodiment 2: PCR obtains gene clone from placenta cDNA:
Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (pharmacia company) of purifying.With MMLV-RT-SuperscriptII (GIBCO BRL), ThermoScript II is carried out reverse transcription reaction at 42 ℃, obtains placenta cDNA.Utilize the different primer of commentaries on classics (as shown in the table) of each gene, by 97 ℃ of 3 ' 1 circulation.94 ℃ 30 " 60 ℃ 30 " 72 ℃ 1 ', 35 circulations, pcr amplification is carried out in 72 ℃ of 10 ' 1 circulations, obtains the amplified production of each gene.Amplified production is through sequence verification, and the sequence that records with embodiment 1 conforms to, and changes amplified production over to host cell with routine techniques subsequently, thereby obtains recombinant protein.
The gene specific primer sequence
Clone's title Special primer 1 (5 ' → 3 ') Special primer 2 (5 ' → 3 ')
PP4277 TCACGGTCCCACTCAAGTCCTGC TCTGGACACAGAACGAGCAGCCC
PP4377 GGCCCTGGGTTCACCTGATTGAG GAGCCCTCCTCCTAGGGCTGGAA
PP4522 TCCCTGTCCCTGCTTCTCTTCGG AAGCAGAGGGCGAAAGCTGCAAG
PP4583 GCGCGTCACCAGAGTCGTTTCTC GGTCTTTCCACTTCGGGCTTCTG
PP4664 CAACGCGGACTTCATCATCCCTG CCACCTGATGCAGGCCAACCATA
PP4748 CGACGCGCTGTCTGGAGTCGTAG TCTGCTGACATGAGGACCTGGGG
PP4762 TGAATTGGCGCTATGTCTGGGGA TCAAGCACTTGGCTTCTGCTGCC
PP5242 GGCTGGGTCCCACCAGTGACAAG CGACCTGCAGACGGGACATGACT
PP5656 AGAGCTCTTTGTGTGCCCCAGCC AAGACCCCACCCCACAGATGCTC
Embodiment 3:cDNA cloned sequence is analyzed
1.PP4277 albumen
A: nucleotide sequence (SEQ ID NO:1) length: 1915bp
CACTAAGGCG CAAGGCAGCT GCACATCCAC GGCCAGCCTC GTTGTCTTCA GGGATTTGTT 60
CAAAGTTCAT TCTGAATTGT AAGGTAACCA CTGCAGAACA CGAGCCTTTC CTGGGTATGT 120
GTGGTTATTT CTGCCCTTCA CGGTCCCACT CAAGTCCTGC CTCCCTCAGG GAGACCTCCC 180
AGACCTCACC AAGCCGTGGG CACGTGGACC AGAAGCCGTG GACCCCAGAG GCTCATTTAG 240
GGATCTGCTT TCTGAACCCA GGTCCTAACA TCTGCACTGC AGCGAGCATT TCTGGTGCCT 300
CAAGTGAGTC ACTCCAACTT CCAGCTCACT GAGAGGCGAT GGAGCACTGA GTGCCAGCAT 360
CTTCCAGCAC CAGAGGAGCT GACTCTGAAT AGACAGCTCG GCAGCGGCCT CAGCACAGAG 420
CAGGGGAGCC ACCAGCCACA GGCAGCCACT GAGCCCTTGG CATGTAACCC GGTAACCCAT 480
GCAACAGAAG ACCTGAGCGT CCAGTGTTGC TAGCTTGAAG TAACTTCAGT GGTGTTCCCC 540
AGCGTTTTTC TCATCCCCCT CCCTTCCCTT CCCCTACAGC TTTCTTCCTC TTTCCCCTCC 600
TTCTGAGTCT TTTGTCCCCA GTCCTGGGAA CCTTTTTTCT GACTTGGTGT TTTCTCCTGA 660
ATGCCTCCTT CCTGGCCCCC TAATCAGAAA TGCAGAAGCC ACCTGCCCCA CACAGGGGGC 720
CGCTTTCCCT GCATCTGGCA CTCACCAGGA CAGTCATAGA ACTGGCCTTG GGTGCTTGGG 780
GAGAGTGGAG AGGTCTGGGG GGCATCCACT GGCCTCAGCC TCAGAGCTTC TCATCAGTAA 840
CCCAGGAGGC AAAGGCCCAG CTCGAGATTA GCCCAGGCCA CTGTATCTGT GCCAAGACCT 900
ACAGCTGTCC CTGCAGGGCG TGCATCTCCT GCAGCCCAGG CTATCACAAT GGTGTCCCTT 960
TTGGTGGGGA TGTGAGGCCT GCTGCCTGCC TGGGAAGAAG CTGACAGGAT TGTTGGTGCT 1020
GTGGGGAGAA GCTGGTGTAA CTGAGACCCA TGAGGGAGGC CCTTTGTGGG TCTGTGGCTG 1080
GGAGGAGCTC TGGGGAAGAT GGGCCTTGAG AAATCCCAGC TTAGCACCTT TGACAGTTGC 1140
TCCACTGAGT CACCCTAGAC TGCAATCTCA CAGATGTTCT AACCACCCTC TCCCTGGGGA 1200
GCCCTTGGGG GTGCTGCAAG CCCAGTCCTC ACTCACTGGC TTTGTGCTTC TCTTCCCGAC 1260
TTCCTTTTTT TGCTCCTGTT GAGAAATGTG GGTCCAGGGC TGCGTGCTCC AGTGTGCGGA 1320
AGGAAGGGCA GCGTGCTCTT TGGACTGAGC TGGCTCAAGG GAAGCTGGGT CTGAGGGGAG 1380
ACTGACCTCG ACCCTTCTTA AATGAAACAC CTTTTCCCTG CGTCTCAGTC ACCAGTTACC 1440
TGGCCTGATC ACTGTGTGCC ACACCCCATC ACATCCATCC CCACAAGAAT GCCAGGAGGA 1500
GGGGTCATTG GCCTTTTTGC CAGCACAAAC CTCTGGAGGC CAGCTCCCAA GACCCGCCAG 1560
GCACACCTGC CAGAGTGCCT TAACTTGGGA TGTCTGCAAA CCTCTGTCCA TGGCCGAACC 1620
CACCCCACTG CCTGTTACTG TAAATAAAGG TCTATTAGCA TGCCACCATG CCCCTCCTCC 1680
GATGCATACA GTGCCCAGCT CAGCACCTGG TGCAAAGTAA GCACAATCTG GCTTCCAGCC 1740
ATCACTGCTG TTGCTACTAT CACAAATATT AGGCCCCTTC TAGAATGCAA AGATAACCCA 1800
GGGCTGCTCG TTCTGTGTCC AGATCACAGA GTTTTTCTTT TTATCCTTAT CAACTTTATT 1860
GAGGGATAAT TTACAGGCAA TAAATCATAT CCATTTAAAA AAAAAAAAAA AAAAA 1915
B: aminoacid sequence (SEQ ID NO:2) length: 104 amino acid
1 MQKPPAPHRG PLSLHLALTR TVIELALGAW GEWRGLGGIH WPQPQSFSSV TQEAKAQLEI
61 SPGHCICAKT YSCPCRACIS CSPGYHNGVP FGGDVRPAAC LGRS
C. Nucleotide and amino acid composite sequence (SEQ ID NO:3)
Clone number: PP4277
Start code: 690 ATG stop coding: 1004 TGA
Protein molecular weight: 11076
1 CA CTA AGG CGC AAG GCA GCT GCA CAT CCA CGG CCA GCC TCG TTG TCT 47
48 TCA GGG ATT TGT TCA AAG TTC ATT CTG AAT TGT AAG GTA ACC ACT GCA 95
96 GAA CAC GAG CCT TTC CTG GGT ATG TGT GGT TAT TTC TGC CCT TCA CGG 143
144 TCC CAC TCA AGT CCT GCC TCC CTC AGG GAG ACC TCC CAG ACC TCA CCA 191
192 AGC CGT GGG CAC GTG GAC CAG AAG CCG TGG ACC CCA GAG GCT CAT TTA 239
240 GGG ATC TGC TTT CTG AAC CCA GGT CCT AAC ATC TGC ACT GCA GCG AGC 287
288 ATT TCT GGT GCC TCA AGT GAG TCA CTC CAA CTT CCA GCT CAC TGA GAG 335
336 GCG ATG GAG CAC TGA GTG CCA GCA TCT TCC AGC ACC AGA GGA GCT GAC 383
384 TCT GAA TAG ACA GCT CGG CAG CGG CCT CAG CAC AGA GCA GGG GAG CCA 431
432 CCA GCC ACA GGC AGC CAC TGA GCC CTT GGC ATG TAA CCC GGT AAC CCA 479
480 TGC AAC AGA AGA CCT GAG CGT CCA GTG TTG CTA GCT TGA AGT AAC TTC 527
528 AGT GGT GTT CCC CAG CGT TTT TCT CAT CCC CCT CCC TTC CCT TCC CCT 575
576 ACA GCT TTC TTC CTC TTT CCC CTC CTT CTG AGT CTT TTG TCC CCA GTC 623
624 CTG GGA ACC TTT TTT CTG ACT TGG TGT TTT CTC CTG AAT GCC TCC TTC 671
672 CTG GCC CCC TAA TCA GAA ATG CAG AAG CCA CCT GCC CCA CAC AGG GGG 719
1 Met Gln Lys Pro Pro Ala Pro His Arg Gly 10
720 CCG CTT TCC CTG CAT CTG GCA CTC ACC AGG ACA GTC ATA GAA CTG GCC 767
11 Pro Leu Ser Leu His Leu Ala Leu Thr Arg Thr Val Ile Glu Leu Ala 26
768 TTG GGT GCT TGG GGA GAG TGG AGA GGT CTG GGG GGC ATC CAC TGG CCT 815
27 Leu Gly Ala Trp Gly Glu Trp Arg Gly Leu Gly Gly Ile His Trp Pro 42
816 CAG CCT CAG AGC TTC TCA TCA GTA ACC CAG GAG GCA AAG GCC CAG CTC 863
43 Gln Pro Gln Ser Phe Ser Ser Val Thr Gln Glu Ala Lys Ala Gln Leu 58
864 GAG ATT AGC CCA GGC CAC TGT ATC TGT GCC AAG ACC TAC AGC TGT CCC 911
59 Glu Ile Ser Pro Gly His Cys Ile Cys Ala Lys Thr Tyr Ser Cys Pro 74
912 TGC AGG GCG TGC ATC TCC TGC AGC CCA GGC TAT CAC AAT GGT GTC CCT 959
75 Cys Arg Ala Cys Ile Ser Cys Ser Pro Gly Tyr His Asn Gly Val Pro 90
960 TTT GGT GGG GAT GTG AGG CCT GCT GCC TGC CTG GGA AGA AGC TGA CAG 1007
91 Phe Gly Gly Asp Val Arg Pro Ala Ala Cys Leu Gly Arg Ser *** 105
1008 GAT TGT TGG TGC TGT GGG GAG AAG CTG GTG TAA CTG AGA CCC ATG AGG 1055
1056 GAG GCC CTT TGT GGG TCT GTG GCT GGG AGG AGC TCT GGG GAA GAT GGG 1103
1104 CCT TGA GAA ATC CCA GCT TAG CAC CTT TGA CAG TTG CTC CAC TGA GTC 1151
1152 ACC CTA GAC TGC AAT CTC ACA GAT GTT CTA ACC ACC CTC TCC CTG GGG 1199
1200 AGC CCT TGG GGG TGC TGC AAG CCC AGT CCT CAC TCA CTG GCT TTG TGC 1247
1248 TTC TCT TCC CGA CTT CCT TTT TTT GCT CCT GTT GAG AAA TGT GGG TCC 1295
1296 AGG GCT GCG TGC TCC AGT GTG CGG AAG GAA GGG CAG CGT GCT CTT TGG 1343
1344 ACT GAG CTG GCT CAA GGG AAG CTG GGT CTG AGG GGA GAC TGA CCT CGA 1391
1392 CCC TTC TTA AAT GAA ACA CCT TTT CCC TGC GTC TCA GTC ACC AGT TAC 1439
1440 CTG GCC TGA TCA CTG TGT GCC ACA CCC CAT CAC ATC CAT CCC CAC AAG 1487
1488 AAT GCC AGG AGG AGG GGT CAT TGG CCT TTT TGC CAG CAC AAA CCT CTG 1535
1536 GAG GCC AGC TCC CAA GAC CCG CCA GGC ACA CCT GCC AGA GTG CCT TAA 1583
1584 CTT GGG ATG TCT GCA AAC CTC TGT CCA TGG CCG AAC CCA CCC CAC TGC 1631
1632 CTG TTA CTG TAA ATA AAG GTC TAT TAG CAT GCC ACC ATG CCC CTC CTC 1679
1680 CGA TGC ATA CAG TGC CCA GCT CAG CAC CTG GTG CAA AGT AAG CAC AAT 1727
1728 CTG GCT TCC AGC CAT CAC TGC TGT TGC TAC TAT CAC AAA TAT TAG GCC 1775
1776 CCT TCT AGA ATG CAA AGA TAA CCC AGG GCT GCT CGT TCT GTG TCC AGA 1823
1824 TCA CAG AGT TTT TCT TTT TAT CCT TAT CAA CTT TAT TGA GGG ATA ATT 1871
1872 TAC AGG CAA TAA ATC ATA TCC ATT TAA AAA AAA AAA AAA AAA AA 1915
2.PP4377 albumen
A: nucleotide sequence (SEQ ID NO:4) length: 2132bp
GGACGAGACT TGATGGAGCC ACCTATACAG TGCTGTTCAT TGGCACAGGA GACGGCTGGC 60
TGCTCAAGGC TGTGAGCCTG GGGCCCTGGG TTCACCTGAT TGAGGAGCTG CAGCTGTTTG 120
ACCAGGAGCC CATGAGAAGC CTGGTGCTAT CTCAGAGCAA GAAGCTGCTC TTTGCCGGCT 180
CCCGCTCTCA GCTGGTGCAG CTGCCCGTGG CCGACTGCAT GAAGTATCGC TCCTGTGCAG 240
ACTGTGTCCT CGCCCGGGAC CCCTATTGCG CCTGGAGCGT CAACACCAGC CGCTGTGTGC 300
CATGGGTGGC CACTCTGGAT CTCTACTGAT CCAGCATGTG ATGACCTCGG ACACTTCAGG 360
CATCTGCAAC CTCCGTGGCA GTAAGAAAGT CAGGCCCACT CCCAAAAACA TCACGGTGGT 420
GGCGGGCACA GACCTGGTGC TGCCCTGCCA CCTCTCCTCC AACTTGGCCC ATGCCCGCTG 480
GACCTTTGGG GGCCGGGACC TGCCTGCGGA ACAGCCCGGG TCCTTCCTCT ACGATGCCCG 540
GCTCCAGGCC TGGTTGTGAT GGCTGCCCAG CCCCGCCATG CCGGGGCCTA CCACTGCTTT 600
TCAGAGGAGC AGGGGGCGCG GCTGGCTGCT GAAGGCTACC TTGTGGCTGT CGTGGCAGGC 660
CCTGTCGGTG ACCTTGGAGG CCCGGCCCCC CCTGGAAAAC CTGGGGCTGG TGTGGCTGGC 720
GGTGGTGGCC CTGGGGGCTG TGTGCCTGGT GCTGCTGCTG CTGGTGCTGT CATTGCGCCG 780
GCGGCTGCGG GAAGAGCTGG AGAAAGGGGC CAAGCTACTG AGAGGACCTT GGTGTACCCC 840
CTGGAGCTGC CCAAGGAGCC CACCAGTCCC CCCTTCCGCC CTGTCCTGAA CCAGATGAGA 900
AACTTTGGGA TCCTGTCGTT ACTACTATTC AGATGGCTCC CTTAAGATAG TACCTGGGCA 960
TGCCCGGTGC CAGCCCGGTG GGGGGCCCCC TTCGCCACCT CCAGGCATCC CAGGCCAGCC 1020
TCTGCCTTCT CCAACTCGGC TTCACCTGGG GGGTGGGCGG AACTCAAATG CCAATGGTTA 1080
CGTGCGCTTA CAACTAGGAG GGGAGGACCG GGGAGGGCTC GGGCACCCCC TGCCTGAGCT 1140
CGCGGATGAA CTGAGACGCA AACTGCAGCA ACGCCAGCCA CTGCCCGACT CCAACCCCGA 1200
GGAGTCATCA GTATGAGGGG AACCCCCACC GCGTCGGCGG GAAGCGTGGG AGGTGTAGCT 1260
CCTACTTTTG CACAGGCACC AGCTACCTCA GGGACATGGC ACGGGCACCT GCTCTGTCTG 1320
GGACAGATAC TGCCCAGCAC CCACCCGGCC ATGAGGACCT GCTCTGCTCA GCACGGGCAC 1380
TGACCTTGGT GTGGCTCACC AGGGCACCAG CCTCGCAGAA GGCATCTTCC TCCTCTCTGT 1440
GAATCACAGA CACGCGGGAC CCCAGCCGCC AAAACTTTTC AAGGCAGAAG TTTCAAGATG 1500
TGTGTTTGTC TGTATTTGCA CATGTGTTTG TGTGTGTGTG TATGTGTGTG TCCACGCGCG 1560
TGCGCGCTTG TGGCATAGCC TTCCTGTTTC TGTCAAGTCT TCCCTTGGCC TGGGTCCTCC 1620
TGGTGAGTCA TTGGAGCTAT GAAGGGGAAG GGGTCGTATC ACTTTGTCTC TCCTACCCCC 1680
ACTGCCCCGA GTGTCGGGCA GCGATGTACA TATGGAGGTG GGGTGGACAG GGTGCTGTGC 1740
CCCTTCAGAG GGAGTGCAGG GCTTGGGGTG GGCCTAGTCC TGCTCCTAGG GCTGTGAATG 1800
TTTTCAGGGT GGGGGGAGGG AGATGGAGCC TCCTGTGTGT TTGGGGGGAA GGGTGGGTGG 1860
GGCCTCCCAC TTGGCCCCGG GGTTCAGTGG TATTTTATAC TTGCCTTCTT CCTGTACAGG 1920
GCTGGGAAAA GCTGTGTGAA GGGAGAGAAG GGAAAGGGTG GGCCTGCTGT GGACAATGGC 1980
ATACTCTCTT CCAGCCCTAG GAGGAGGGCT CCTAACAGTG TAACTTATTG TGTCCCCGCG 2040
TATTTATTTG TTGTAAATAT TTGAGTATTT TTATATTGCC AAATAAAATG GGAAAAAAAA 2100
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 2132
B: aminoacid sequence (SEQ ID NO:5) length: 152 amino acid
1 MCVSTRVRAC GIAFLFLSSL PLAWVLLVSH WSYEGEGVVS LCLSYPHCPE CRAAMYIWRW
61 GGQGAVPLQR ECRAWGGPSP APRAVNVFRV GGGRWSLLCV WGEGWVGPPT WPRGSVVFYT
121 CLLPVQGWEK LCEGREGKGW ACCGQWHTLF QP
C. Nucleotide and amino acid composite sequence (SEQ ID NO:6)
Clone number: PP4377
Start code: 1542 ATG stop coding: 2000 TAG
Protein molecular weight: 16914
1 GG ACG AGA CTT GAT GGA GCC ACC TAT ACA GTG CTG TTC ATT GGC ACA 47
48 GGA GAC GGC TGG CTG CTC AAG GCT GTG AGC CTG GGG CCC TGG GTT CAC 95
96 CTG ATT GAG GAG CTG CAG CTG TTT GAC CAG GAG CCC ATG AGA AGC CTG 143
144 GTG CTA TCT CAG AGC AAG AAG CTG CTC TTT GCC GGC TCC CGC TCT CAG 191
192 CTG GTG CAG CTG CCC GTG GCC GAC TGC ATG AAG TAT CGC TCC TGT GCA 239
240 GAC TGT GTC CTC GCC CGG GAC CCC TAT TGC GCC TGG AGC GTC AAC ACC 287
288 AGC CGC TGT GTG CCA TGG GTG GCC ACT CTG GAT CTC TAC TGA TCC AGC 335
336 ATG TGA TGA CCT CGG ACA CTT CAG GCA TCT GCA ACC TCC GTG GCA GTA 383
384 AGA AAG TCA GGC CCA CTC CCA AAA ACA TCA CGG TGG TGG CGG GCA CAG 431
432 ACC TGG TGC TGC CCT GCC ACC TCT CCT CCA ACT TGG CCC ATG CCC GCT 479
480 GGA CCT TTG GGG GCC GGG ACC TGC CTG CGG AAC AGC CCG GGT CCT TCC 527
528 TCT ACG ATG CCC GGC TCC AGG CCT GGT TGT GAT GGC TGC CCA GCC CCG 575
576 CCA TGC CGG GGC CTA CCA CTG CTT TTC AGA GGA GCA GGG GGC GCG GCT 623
624 GGC TGC TGA AGG CTA CCT TGT GGC TGT CGT GGC AGG CCC TGT CGG TGA 671
672 CCT TGG AGG CCC GGC CCC CCC TGG AAA ACC TGG GGC TGG TGT GGC TGG 719
720 CGG TGG TGG CCC TGG GGG CTG TGT GCC TGG TGC TGC TGC TGC TGG TGC 767
768 TGT CAT TGC GCC GGC GGC TGC GGG AAG AGC TGG AGA AAG GGG CCA AGC 815
816 TAC TGA GAG GAC CTT GGT GTA CCC CCT GGA GCT GCC CAA GGA GCC CAC 863
864 CAG TCC CCC CTT CCG CCC TGT CCT GAA CCA GAT GAG AAA CTT TGG GAT 911
912 CCT GTC GTT ACT ACT ATT CAG ATG GCT CCC TTA AGA TAG TAC CTG GGC 959
960 ATG CCC GGT GCC AGC CCG GTG GGG GGC CCC CTT CGC CAC CTC CAG GCA 1007
1008 TCC CAG GCC AGC CTC TGC CTT CTC CAA CTC GGC TTC ACC TGG GGG GTG 1055
1056 GGC GGA ACT CAA ATG CCA ATG GTT ACG TGC GCT TAC AAC TAG GAG GGG 1103
1104 AGG ACC GGG GAG GGC TCG GGC ACC CCC TGC CTG AGC TCG CGG ATG AAC 1151
1152 TGA GAC GCA AAC TGC AGC AAC GCC AGC CAC TGC CCG ACT CCA ACC CCG 1199
1200 AGG AGT CAT CAG TAT GAG GGG AAC CCC CAC CGC GTC GGC GGG AAG CGT 1247
1248 GGG AGG TGT AGC TCC TAC TTT TGC ACA GGC ACC AGC TAC CTC AGG GAC 1295
1296 ATG GCA CGG GCA CCT GCT CTG TCT GGG ACA GAT ACT GCC CAG CAC CCA 1343
1344 CCC GGC CAT GAG GAC CTG CTC TGC TCA GCA CGG GCA CTG ACC TTG GTG 1391
1392 TGG CTC ACC AGG GCA CCA GCC TCG CAG AAG GCA TCT TCC TCC TCT CTG 1439
1440 TGA ATC ACA GAC ACG CGG GAC CCC AGC CGC CAA AAC TTT TCA AGG CAG 1487
1488 AAG TTT CAA GAT GTG TGT TTG TCT GTA TTT GCA CAT GTG TTT GTG TGT 1535
1536 GTG TGT ATG TGT GTG TCC ACG CGC GTG CGC GCT TGT GGC ATA GCC TTC 1583
1 Met Cys Val Ser Thr Arg Val Arg Ala Cys Gly Ile Ala Phe 14
1584 CTG TTT CTG TCA AGT CTT CCC TTG GCC TGG GTC CTC CTG GTG AGT CAT 1631
15 Leu Phe Leu Ser Ser Leu Pro Leu Ala Trp Val Leu Leu Val Ser His 30
1632 TGG AGC TAT GAA GGG GAA GGG GTC GTA TCA CTT TGT CTC TCC TAC CCC 1679
31 Trp Ser Tyr Glu Gly Glu Gly Val Val Ser Leu Cys Leu Ser Tyr Pro 46
1680 CAC TGC CCC GAG TGT CGG GCA GCG ATG TAC ATA TGG AGG TGG GGT GGA 1727
47 His Cys Pro Glu Cys Arg Ala Ala Met Tyr Ile Trp Arg Trp Gly Gly 62
1728 CAG GGT GCT GTG CCC CTT CAG AGG GAG TGC AGG GCT TGG GGT GGG CCT 1775
63 Gln Gly Ala Val Pro Leu Gln Arg Glu Cys Arg Ala Trp Gly Gly Pro 78
1776 AGT CCT GCT CCT AGG GCT GTG AAT GTT TTC AGG GTG GGG GGA GGG AGA 1823
79 Ser Pro Ala Pro Arg Ala Val Asn Val Phe Arg Val Gly Gly Gly Arg 94
1824 TGG AGC CTC CTG TGT GTT TGG GGG GAA GGG TGG GTG GGG CCT CCC ACT 1871
95 Trp Ser Leu Leu Cys Val Trp Gly Glu Gly Trp Val Gly Pro Pro Thr 110
1872 TGG CCC CGG GGT TCA GTG GTA TTT TAT ACT TGC CTT CTT CCT GTA CAG 1919
111 Trp Pro Arg Gly Ser Val Val Phe Tyr Thr Cys Leu Leu Pro Val Gln 126
1920 GGC TGG GAA AAG CTG TGT GAA GGG AGA GAA GGG AAA GGG TGG GCC TGC 1967
127 Gly Trp Glu Lys Leu Cys Glu Gly Arg Glu Gly Lys Gly Trp Ala Cys 142
1968 TGT GGA CAA TGG CAT ACT CTC TTC CAG CCC TAG GAG GAG GGC TCC TAA 2015
143 Cys Gly Gln Trp His Thr Leu Phe Gln Pro *** 153
2016 CAG TGT AAC TTA TTG TGT CCC CGC GTA TTT ATT TGT TGT AAA TAT TTG 2063
2064 AGT ATT TTT ATA TTG CCA AAT AAA ATG GGA AAA AAA AAA AAA AAA AAA 2111
2112 AAA AAA AAA AAA AAA AAA AAA 2132
3.PP4522 albumen
A: nucleotide sequence (SEQ ID NO:7) length: 2035bp
GGCACCTCGA GTTTTTTTTT TTTTTATTCC CAGGACATCA AGGAGACTTT CAATAGGTGT 60
GTTCCCAGTC TTTGCCCTTC GTGACCCAGT GGCATCTGGT TCCCTGTCCC TGCTTCTCTT 120
CGGTATCCCT CTCCTCTCCT TCCTTCCCCA GGACCTGAGT TTCCATCTCC TGGACCCTCC 180
TCTCCTTCCC CTCAGCTTTT GCTTTTCCCT CTGGGAATAT CGTGGTCCCA CCCCCTGCCC 240
GGTCCCCTTC CTCCAGGTGT GAAGAGGTAC AGCTGCAGCC CCCAGAGGTC TGGTCCCCTG 300
ACCCGTGCCA ACCCCATAGC CATGACTTCC TGACAGATGC CATCGTGAGG AAAATGAGCC 360
GGATGTTCTG TCAGGCTGCG AGAGTGGACC TGACGCTGGA CCCTGACACG GCTCACCCGG 420
CCCTGATGCT GTCCCCTGAC CGCCGGGGGG TCCGCCTGGC AGAGCGGCGG CAGGAGGTTG 480
CTGACCATCC CAAGCGCTTC TCGGCCGACT GCTGCGTACT GGGGGCCCAG GGCTTCCGCT 540
CCGGCCGGCA CTACTGGGAG GTAGAGGTGG GCGGGCGGCG GGGCTGGGCG GTGGGTGCTG 600
CCCGTGAATC AACCCATCAT AAGGAAAAGG TGGGCCCTGG GGGTTCCTCC GTGGGCAGCG 660
GGGATGCCAG CTCCTCGCGC CATCACCATC GCCGCCGCCG GCTCCACCTG CCCCAGCAGC 720
CCCTGCTCCA GCGGGAAGTG TGGTGCGTGG GCACCAACGG CAAACGCTAT CAGGCCCAGA 780
GCTCCACAGA ACAGACGCTG CTGAGCCCCA GTGAGAAACC AAGGCGCTTT GGTGTGTACC 840
TGGACTATGA AGCTGGGCGC CTGGGCTTCT ACAACGCAGA GACTCTAGCC CACGTGCACA 900
CCTTCTCGGC TGCCTTCCTG GGCGAGCGTG TCTTTCCTTT CTTCCGGGTG CTCTCCAAGG 960
GCACCCGCAT CAAGCTCTGC CCTTGATTAT CCTGCCACCC GCAGGGGCCC CTCTGTCAGC 1020
ACTTGGGGGG TGGGTGGTGG AGGGTGGCCC GTAAGTTTGA GGGCTCAAAG GCTCTTCCCA 1080
CTGCTTGTTA CTGTGTTGCT TCCCACTCCC CCTTGACCCC AGGCCCCTGC TTCTCCCTCT 1140
AGGAGCCTAA AGAACCCTCC TGGCCTCCAG CTCAGCCTTC TCTCACCTAC TATGTCTGTC 1200
CAACAGGTCT GCATGGGTCC CTGATAATGA GAACAGCTGC CTGGTCTTCT CTCCCAGTCT 1260
GCCTAGCCCA GCCCTGGGAC TGGAATTTGA GTAGGGGATG AGGGGAAATT GTAATTTCAT 1320
TCCTTAACTT CCTTTTCCCC ACCCCTGCTC TTCAACCTCT TTATCAGTTC TGAGGCTGGA 1380
GGGTTTGGGC AAGGCAACAT CCCCATTCCA ATTCCATTTT CTGATGCAGA TTTTAGCTGA 1440
GGGATTTGGA AGCCATTTGG GGAGGCAGGC TGGGCCAAAG GGTAGAGCTG GGTAATAAAT 1500
GTCTATTCTC CTGGGGAGGA GGGATTCTAA ACTTTCCTTC CGTCCTCAAT TTCTACCTCC 1560
ATAGACCGGC CAGAATTTAG CTTCACTTGA GAGAGATCTG GAATGGTCGC CATGATTGAA 1620
ACCACGCACC ATTACATCAT CATTACATTA ATTACATCAA CATAAATTAT TTCTTCCCCC 1680
TTCCCTTTTC CAGCACTCAA CCAAGGAGCA AAGCTCATCC CACCCCACAC CCCTCCCAGG 1740
TCTGCTCACT GCCAGGCTCC TCTCCCCTTT GTTCAGTGGA GCTGGCTTTT CTCCCAGCCC 1800
CTTTCCATGC CTTTCACTCC ATTTGGCAAG CTCTGAGGGG GAGCCTGGGG ACGGGTTTGG 1860
GTCCCCAGGA GGAGAGCCTT GGGTATAATC TATTTTTCTA GGAGCCTCTT GCCTTGTCAC 1920
TTGCAGCTTT CGCCCTCTGC TTTGATGGCT GAGGTGAACT CATGTTCTTT GGGAAAAGGG 1980
AAGGCGTGCT GTGGAAATAA AATGTTTATT TGCTTCTAAA AAAAAAAAAA AAAAA 2035
B: aminoacid sequence (SEQ ID NO:8) length: 210 amino acid
1 MSRMFCQAAR VDLTLDPDTA HPALMLSPDR RGVRLAERRQ EVADHPKRFS ADCCVLGAQG
61 FRSGRHYWEV EVGGRRGWAV GAARESTHHK EKVGPGGSSV GSGDASSSRH HHRRRRLHLP
121 QQPLLQREVW CVGTNGKRYQ AQSSTEQTLL SPSEKPRRFG VYLDYEAGRL GFYNAETLAH
181 VHTFSAAFLG ERVFPFFRVL SKGTRIKLCP
C. Nucleotide and amino acid composite sequence (SEQ ID NO:9)
Clone number: PP4522
Start code: 354 ATG stop coding: 986 TGA
Protein molecular weight: 23607
1 GG CAC CTC GAG TTT TTT TTT TTT TTA TTC CCA GGA CAT CAA GGA GAC 47
48 TTT CAA TAG GTG TGT TCC CAG TCT TTG CCC TTC GTG ACC CAG TGG CAT 95
96 CTG GTT CCC TGT CCC TGC TTC TCT TCG GTA TCC CTC TCC TCT CCT TCC 143
144 TTC CCC AGG ACC TGA GTT TCC ATC TCC TGG ACC CTC CTC TCC TTC CCC 191
192 TCA GCT TTT GCT TTT CCC TCT GGG AAT ATC GTG GTC CCA CCC CCT GCC 239
240 CGG TCC CCT TCC TCC AGG TGT GAA GAG GTA CAG CTG CAG CCC CCA GAG 287
288 GTC TGG TCC CCT GAC CCG TGC CAA CCC CAT AGC CAT GAC TTC CTG ACA 335
336 GAT GCC ATC GTG AGG AAA ATG AGC CGG ATG TTC TGT CAG GCT GCG AGA 383
1 Met Ser Arg Met Phe Cys Gln Ala Ala Arg 10
384 GTG GAC CTG ACG CTG GAC CCT GAC ACG GCT CAC CCG GCC CTG ATG CTG 431
11 Val Asp Leu Thr Leu Asp Pro Asp Thr Ala His Pro Ala Leu Met Leu 26
432 TCC CCT GAC CGC CGG GGG GTC CGC CTG GCA GAG CGG CGG CAG GAG GTT 479
27 Ser Pro Asp Arg Arg Gly Val Arg Leu Ala Glu Arg Arg Gln Glu Val 42
480 GCT GAC CAT CCC AAG CGC TTC TCG GCC GAC TGC TGC GTA CTG GGG GCC 527
43 Ala Asp His Pro Lys Arg Phe Ser Ala Asp Cys Cys Val Leu Gly Ala 58
528 CAG GGC TTC CGC TCC GGC CGG CAC TAC TGG GAG GTA GAG GTG GGC GGG 575
59 Gln Gly Phe Arg Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Gly 74
576 CGG CGG GGC TGG GCG GTG GGT GCT GCC CGT GAA TCA ACC CAT CAT AAG 623
75 Arg Arg Gly Trp Ala Val Gly Ala Ala Arg Glu Ser Thr His His Lys 90
624 GAA AAG GTG GGC CCT GGG GGT TCC TCC GTG GGC AGC GGG GAT GCC AGC 671
91 Glu Lys Val Gly Pro Gly Gly Ser Ser Val Gly Ser Gly Asp Ala Ser 106
672 TCC TCG CGC CAT CAC CAT CGC CGC CGC CGG CTC CAC CTG CCC CAG CAG 719
107 Ser Ser Arg His His His Arg Arg Arg Arg Leu His Leu Pro Gln Gln 122
720 CCC CTG CTC CAG CGG GAA GTG TGG TGC GTG GGC ACC AAC GGC AAA CGC 767
123 Pro Leu Leu Gln Arg Glu Val Trp Cys Val Gly Thr Asn Gly Lys Arg 138
768 TAT CAG GCC CAG AGC TCC ACA GAA CAG ACG CTG CTG AGC CCC AGT GAG 815
139 Tyr Gln Ala Gln Ser Ser Thr Glu Gln Thr Leu Leu Ser Pro Ser Glu 154
816 AAA CCA AGG CGC TTT GGT GTG TAC CTG GAC TAT GAA GCT GGG CGC CTG 863
155 Lys Pro Arg Arg Phe Gly Val Tyr Leu Asp Tyr Glu Ala Gly Arg Leu 170
864 GGC TTC TAC AAC GCA GAG ACT CTA GCC CAC GTG CAC ACC TTC TCG GCT 911
171 Gly Phe Tyr Asn Ala Glu Thr Leu Ala His Val His Thr Phe Ser Ala 186
912 GCC TTC CTG GGC GAG CGT GTC TTT CCT TTC TTC CGG GTG CTC TCC AAG 959
187 Ala Phe Leu Gly Glu Arg Val Phe Pro Phe Phe Arg Val Leu Ser Lys 202
960 GGC ACC CGC ATC AAG CTC TGC CCT TGA TTA TCC TGC CAC CCG CAG GGG 1007
203 Gly Thr Arg Ile Lys Leu Cys Pro *** 211
1008 CCC CTC TGT CAG CAC TTG GGG GGT GGG TGG TGG AGG GTG GCC CGT AAG 1055
1056 TTT GAG GGC TCA AAG GCT CTT CCC ACT GCT TGT TAC TGT GTT GCT TCC 1103
1104 CAC TCC CCC TTG ACC CCA GGC CCC TGC TTC TCC CTC TAG GAG CCT AAA 1151
1152 GAA CCC TCC TGG CCT CCA GCT CAG CCT TCT CTC ACC TAC TAT GTC TGT 1199
1200 CCA ACA GGT CTG CAT GGG TCC CTG ATA ATG AGA ACA GCT GCC TGG TCT 1247
1248 TCT CTC CCA GTC TGC CTA GCC CAG CCC TGG GAC TGG AAT TTG AGT AGG 1295
1296 GGA TGA GGG GAA ATT GTA ATT TCA TTC CTT AAC TTC CTT TTC CCC ACC 1343
1344 CCT GCT CTT CAA CCT CTT TAT CAG TTC TGA GGC TGG AGG GTT TGG GCA 1391
1392 AGG CAA CAT CCC CAT TCC AAT TCC ATT TTC TGA TGC AGA TTT TAG CTG 1439
1440 AGG GAT TTG GAA GCC ATT TGG GGA GGC AGG CTG GGC CAA AGG GTA GAG 1487
1488 CTG GGT AAT AAA TGT CTA TTC TCC TGG GGA GGA GGG ATT CTA AAC TTT 1535
1536 CCT TCC GTC CTC AAT TTC TAC CTC CAT AGA CCG GCC AGA ATT TAG CTT 1583
1584 CAC TTG AGA GAG ATC TGG AAT GGT CGC CAT GAT TGA AAC CAC GCA CCA 1631
1632 TTA CAT CAT CAT TAC ATT AAT TAC ATC AAC ATA AAT TAT TTC TTC CCC 1679
1680 CTT CCC TTT TCC AGC ACT CAA CCA AGG AGC AAA GCT CAT CCC ACC CCA 1727
1728 CAC CCC TCC CAG GTC TGC TCA CTG CCA GGC TCC TCT CCC CTT TGT TCA 1775
1776 GTG GAG CTG GCT TTT CTC CCA GCC CCT TTC CAT GCC TTT CAC TCC ATT 1823
1824 TGG CAA GCT CTG AGG GGG AGC CTG GGG ACG GGT TTG GGT CCC CAG GAG 1871
1872 GAG AGC CTT GGG TAT AAT CTA TTT TTC TAG GAG CCT CTT GCC TTG TCA 1919
1920 CTT GCA GCT TTC GCC CTC TGC TTT GAT GGC TGA GGT GAA CTC ATG TTC 1967
1968 TTT GGG AAA AGG GAA GGC GTG CTG TGG AAA TAA AAT GTT TAT TTG CTT 2015
2016 CTA AAA AAA AAA AAA AAA AA 2035
4.PP4583 albumen
A: nucleotide sequence (SEQ ID NO:10) length: 2142bp
CCGAAACGGG GAAGTTGTCT TGTGTGGAGA GGTTAGTAGA GCAGCGCGCG CGTCACCAGA 60
GTCGTTTCTC TTCGGAGTCT TAGGTGATCG AGGGTGTGCC CAGGGGGCGG GACTTGTTTG 120
CGCCTCCCGT TCCCTCCCAA TTTCCAAACG TGTCACCCCG GCGCCGACGG CCCTGTGCAG 180
GGGAAGCAGA TGGAGTTCAA GCTGGAGGCT CATCGCATCG TCAGCATCTC TCTGGGCAAG 240
ATCTACAACT CGCGGGTCCA GCGCGGCGGC ATCAAGCTGC ATAAGAACCT CCTGGTCTCG 300
CTGGTGCTGC GCAGCGCCCG CCAAGTCTAC CTGAGCGACC CGTGCCCCGG CCTCTACCTG 360
GCCGGTCCCG CTGGGACCCC GGCGCCGCCA CCGCAGCAGC AGCCCGGGGA GCCGGCGGCC 420
GGGCCACCCG CCGGCTGGGG AGAGCCGCCC CCGCCCGCCG CTCATGCCTC TTGGCCGGAG 480
ACCGAGCCGC AGCCGGAGCG CTCCTCCGTC TCAGACGCGC CGCGGGTAGG GGACGAGGTG 540
CCGGTGGCCA CGGTGACTGG AGTCGGGGAC GTTTTTCAGG GCGGAGAGGC GGACGCGACG 600
GAAGCTGCCT GGAGCCGCGT GGAGGGGCCG CGCCAGGCGG CGGCCAGAGA AGCCGAGGGT 660
ACCGCCGGAG GCTGGGGCGT CTTCCCCGAG GTATCTCGTG CCGCGCGCCG CCCCTGCGGC 720
TGCCCCCTAG GCGGGGAGGA CCCGCCGGGT ACACCGGCCG CGACCCCCGG CGCTGCCTGC 780
TGCTGCGCGC CGCGACCAGC GGAGGACGAG CCCCCCGCGC CGCCCGCGGT GTGCCCCAGG 840
AAGCGCTGCG CGGCGGGGGT GGGCGGCGGC CCAGCGGGCT GCCCGGCGCC CGGCTCGACC 900
CCGCTCAAGA AGCCCCGCCG GAACTTAGAG CAGCCGCCGA GTGGAGGAGA GGACGACGAC 960
GCGGAGGAGA TGGAGACCGG GAACGTGGCT AACCTCATCA GCATCTTCGG TTCCAGTTTC 1020
TCGGGACTCC TACGGAAAAG CCCCGGGGGC GGCAGAGAGG AAGAGGAGGG AGAGGAGAGC 1080
GGTCCGGAAG CCGCCGAGCC CGGGCAGATC TGCTGCGATA AGCCGGTGCT GAGAGACATG 1140
AACCCCTGGA GCACAGCCAT CGTGGCCTTC TGAGCCCTTG GCCCCCCTGC GGGGAGGAGG 1200
TGGAGCAGCG GGCGTCCCCG AAGTGAGGGC CAGGCCCTTC CCGGCTGCGA GGACGCCCAG 1260
AGACCGCGGG CGCTGAGCGC GTTGGGCAGA CTGGTTGCCT CTGGGCATCG CAAACTGCCC 1320
CCGGGCGCAT CTGGCTTATC TGGAGACCTG GGAGCTTGAG GCTCATTGGA AGAGGACGAT 1380
CGTTAACTTT TGTGTTTGTG TATGTCTGTG TATGTAAGTT TGTCTTGTGG CATTAAGACT 1440
ATTTTGCTCT TCTGGAATGC ACCACTCCTT CCCCAGGGTT TAAGAGATTG GGGGCAGACA 1500
GGGACTTTCC TCTGCCGGCT GCGTGGAGAA GGAAGCGGCT AAAAGGTTTG GGCCGGGGAG 1560
TTCCCCATTC GTTCTCCGGG GAGGGAGGGA CTTTACACCT ACCCCTCACC GGAAAGCTAG 1620
ACCCGCTTCA GGGCCAGGAG TGGCGTTTCC GCACAGGATT TCCTAAGACG AGAGGGATTT 1680
AGCCAAGAGC ACAGACTTGG ATTCCTTCTG TCCCTCCCCA CCTTTCTCTC CCAATGAAGA 1740
AACGTTTATA TCCTGTGATT ACTCTAGGAA AGGGGCGTAT TAACGGTCCT TTGCTTCCCC 1800
GCAGGGGAGA AAAAGCTTGA CGAACTTCAC AGAAGAGTTC AAGCTTGGAA ATAATGGAGT 1860
TTATAGAGAA AAGATATATT TTTAAAAGCT CTAGGTCAGA AGTACTACAC AGTGCTTTTA 1920
AAAAGTGTTT AATGAGAGTT TACAGACAGA AGCCCGAAGT GGAAAGACCT TATGGTTTTG 1980
TAGATATGGT GACTCCAGTC TTTGTTGTAT AAAGGTTGGG GGAGCTGATA AGGTTTTTGT 2040
ACAGTATTTT CTCCTTCGTT GTATTGATTT TTGTATAAAA ATGTAACTTC TACGTGTCTA 2100
ACACGTATTA AATATTTTGA AGCAAAAAAA AAAAAAAAAA AA 2142
B: aminoacid sequence (SEQ ID NO:11) length: 327 amino acid
1 MEFKLEAHRI VSISLGKIYN SRVQRGGIKL HKNLLVSLVL RSARQVYLSD PCPGLYLAGP
61 AGTPAPPPQQ QPGEPAAGPP AGWGEPPPPA AHASWPETEP QPERSSVSDA PRVGDEVPVA
121 TVTGVGDVFQ GGEADATEAA WSRVEGPRQA AAREAEGTAG GWGVFPEVSR AARRPCGCPL
181 GGEDPPGTPA ATPGAACCCA PRPAEDEPPA PPAVCPRKRC AAGVGGGPAG CPAPGSTPLK
241 KPRRNLEQPP SGGEDDDAEE METGNVANLI SIFGSSFSGL LRKSPGGGRE EEEGEESGPE
301 AAEPGQICCD KPVLRDMNPW STAIVAF
C. Nucleotide and amino acid composite sequence (SEQ ID NO:12)
Clone number: PP4583
Start code: 190 ATG stop coding: 1173 TGA
Protein molecular weight: 33612
1 CCG AAA CGG GGA AGT TGT CTT GTG TGG AGA GGT TAG TAG AGC AGC GCG 48
49 CGC GTC ACC AGA GTC GTT TCT CTT CGG AGT CTT AGG TGA TCG AGG GTG 96
97 TGC CCA GGG GGC GGG ACT TGT TTG CGC CTC CCG TTC CCT CCC AAT TTC 144
145 CAA ACG TGT CAC CCC GGC GCC GAC GGC CCT GTG CAG GGG AAG CAG ATG 192
1 Met 1
193 GAG TTC AAG CTG GAG GCT CAT CGC ATC GTC AGC ATC TCT CTG GGC AAG 240
2 Glu Phe Lys Leu Glu Ala His Arg Ile Val Ser Ile Ser Leu Gly Lys 17
241 ATC TAC AAC TCG CGG GTC CAG CGC GGC GGC ATC AAG CTG CAT AAG AAC 288
18 Ile Tyr Asn Ser Arg Val Gln Arg Gly Gly Ile Lys Leu His Lys Asn 33
289 CTC CTG GTC TCG CTG GTG CTG CGC AGC GCC CGC CAA GTC TAC CTG AGC 336
34 Leu Leu Val Ser Leu Val Leu Arg Ser Ala Arg Gln Val Tyr Leu Ser 49
337 GAC CCG TGC CCC GGC CTC TAC CTG GCC GGT CCC GCT GGG ACC CCG GCG 384
50 Asp Pro Cys Pro Gly Leu Tyr Leu Ala Gly Pro Ala Gly Thr Pro Ala 65
385 CCG CCA CCG CAG CAG CAG CCC GGG GAG CCG GCG GCC GGG CCA CCC GCC 432
66 Pro Pro Pro Gln Gln Gln Pro Gly Glu Pro Ala Ala Gly Pro Pro Ala 81
433 GGC TGG GGA GAG CCG CCC CCG CCC GCC GCT CAT GCC TCT TGG CCG GAG 480
82 Gly Trp Gly Glu Pro Pro Pro Pro Ala Ala His Ala Ser Trp Pro Glu 97
481 ACC GAG CCG CAG CCG GAG CGC TCC TCC GTC TCA GAC GCG CCG CGG GTA 528
98 Thr Glu Pro Gln Pro Glu Arg Ser Ser Val Ser Asp Ala Pro Arg Val 113
529 GGG GAC GAG GTG CCG GTG GCC ACG GTG ACT GGA GTC GGG GAC GTT TTT 576
114 Gly Asp Glu Val Pro Val Ala Thr Val Thr Gly Val Gly Asp Val Phe 129
577 CAG GGC GGA GAG GCG GAC GCG ACG GAA GCT GCC TGG AGC CGC GTG GAG 624
130 Gln Gly Gly Glu Ala Asp Ala Thr Glu Ala Ala Trp Ser Arg Val Glu 145
625 GGG CCG CGC CAG GCG GCG GCC AGA GAA GCC GAG GGT ACC GCC GGA GGC 672
146 Gly Pro Arg Gln Ala Ala Ala Arg Glu Ala Glu Gly Thr Ala Gly Gly 161
673 TGG GGC GTC TTC CCC GAG GTA TCT CGT GCC GCG CGC CGC CCC TGC GGC 720
162 Trp Gly Val Phe Pro Glu Val Ser Arg Ala Ala Arg Arg Pro Cys Gly 177
721 TGC CCC CTA GGC GGG GAG GAC CCG CCG GGT ACA CCG GCC GCG ACC CCC 768
178 Cys Pro Leu Gly Gly Glu Asp Pro Pro Gly Thr Pro Ala Ala Thr Pro 193
769 GGC GCT GCC TGC TGC TGC GCG CCG CGA CCA GCG GAG GAC GAG CCC CCC 816
194 Gly Ala Ala Cys Cys Cys Ala Pro Arg Pro Ala Glu Asp Glu Pro Pro 209
817 GCG CCG CCC GCG GTG TGC CCC AGG AAG CGC TGC GCG GCG GGG GTG GGC 864
210 Ala Pro Pro Ala Val Cys Pro Arg Lys Arg Cys Ala Ala Gly Val Gly 225
865 GGC GGC CCA GCG GGC TGC CCG GCG CCC GGC TCG ACC CCG CTC AAG AAG 912
226 Gly Gly Pro Ala Gly Cys Pro Ala Pro Gly Ser Thr Pro Leu Lys Lys 241
913 CCC CGC CGG AAC TTA GAG CAG CCG CCG AGT GGA GGA GAG GAC GAC GAC 960
242 Pro Arg Arg Asn Leu Glu Gln Pro Pro Ser Gly Gly Glu Asp Asp Asp 257
961 GCG GAG GAG ATG GAG ACC GGG AAC GTG GCT AAC CTC ATC AGC ATC TTC 1008
258 Ala Glu Glu Met Glu Thr Gly Asn Val Ala Asn Leu Ile Ser Ile Phe 273
1009 GGT TCC AGT TTC TCG GGA CTC CTA CGG AAA AGC CCC GGG GGC GGC AGA 1056
274 Gly Ser Ser Phe Ser Gly Leu Leu Arg Lys Ser Pro Gly Gly Gly Arg 289
1057 GAG GAA GAG GAG GGA GAG GAG AGC GGT CCG GAA GCC GCC GAG CCC GGG 1104
290 Glu Glu Glu Glu Gly Glu Glu Ser Gly Pro Glu Ala Ala Glu Pro Gly 305
1105 GAG ATC TGC TGC GAT AAG CCG GTG CTG AGA GAC ATG AAC CCC TGG AGC 1152
306 Gln Ile Cys Cys Asp Lys Pro Val Leu Arg Asp Met Asn Pro Trp Ser 321
1153 ACA GCC ATC GTG GCC TTC TGA GCC CTT GGC CCC CCT GCG GGG AGG AGG 1200
322 Thr Ala Ile Val Ala Phe *** 328
1201 TGG AGC AGC GGG CGT CCC CGA AGT GAG GGC CAG GCC CTT CCC GGC TGC 1248
1249 GAG GAC GCC CAG AGA CCG CGG GCG CTG AGC GCG TTG GGC AGA CTG GTT 1296
1297 GCC TCT GGG CAT CGC AAA CTG CCC CCG GGC GCA TCT GGC TTA TCT GGA 1344
1345 GAC CTG GGA GCT TGA GGC TCA TTG GAA GAG GAC GAT CGT TAA CTT TTG 1392
1393 TGT TTG TGT ATG TCT GTG TAT GTA AGT TTG TCT TGT GGC ATT AAG ACT 1440
1441 ATT TTG CTC TTC TGG AAT GCA CCA CTC CTT CCC CAG GGT TTA AGA GAT 1488
1489 TGG GGG CAG ACA GGG ACT TTC CTC TGC CGG CTG CGT GGA GAA GGA AGC 1536
1537 GGC TAA AAG GTT TGG GCC GGG GAG TTC CCC ATT CGT TCT CCG GGG AGG 1584
1585 GAG GGA CTT TAC ACC TAC CCC TCA CCG GAA AGC TAG ACC CGC TTC AGG 1632
1633 GCC AGG AGT GGC GTT TCC GCA CAG GAT TTC CTA AGA CGA GAG GGA TTT 1680
1681 AGC CAA GAG CAC AGA CTT GGA TTC CTT CTG TCC CTC CCC ACC TTT CTC 1728
1729 TCC CAA TGA AGA AAC GTT TAT ATC CTG TGA TTA CTC TAG GAA AGG GGC 1776
1777 GTA TTA ACG GTC CTT TGC TTC CCC GCA GGG GAG AAA AAG CTT GAC GAA 1824
1825 CTT CAC AGA AGA GTT CAA GCT TGG AAA TAA TGG AGT TTA TAG AGA AAA 1872
1873 GAT ATA TTT TTA AAA GCT CTA GGT CAG AAG TAC TAC ACA GTG CTT TTA 1920
1921 AAA AGT GTT TAA TGA GAG TTT ACA GAC AGA AGC CCG AAG TGG AAA GAC 1968
1969 CTT ATG GTT TTG TAG ATA TGG TGA CTC CAG TCT TTG TTG TAT AAA GGT 2016
2017 TGG GGG AGC TGA TAA GGT TTT TGT ACA GTA TTT TCT CCT TCG TTG TAT 2064
2065 TGA TTT TTG TAT AAA AAT GTA ACT TCT ACG TGT CTA ACA CGT ATT AAA 2112
2113 TAT TTT GAA GCA AAA AAA AAA AAA AAA AAA 2142
5.PP4664 albumen
A: nucleotide sequence (SEQ ID NO:13) length: 2158bp
GCCATCCCTA AGCAGACCCC AGTCCAATAC CTGCTCCCTG AGGCCAAGGC CCAGGACTCA 60
GACAAGATCT GCGTGGTCAT CGACCTGGAC GAGACCCTGG TGCACAGCTC CTTCAAGCCA 120
GTGAACAACG CGGACTTCAT CATCCCTGTG GAGATTGATG GGGTGGTCCA CCAGGTCTAC 180
GTGTTGAAGC GTCCTCATGT GGATGAGTTC CTGCAGCGAA TGGGCGAGCT CTTTGAATGT 240
GTGCTGTTCA CTGCTAGCCT CGCCAAGTAC GCAGACCCAG TAGCTGACCT GCTGGACAAA 300
TGGGGGGCCT TCCGGGCCCG GCTGTTTCGA GAGTCCTGCG TCTTCCACCG GGGGAACTAC 360
GTGAAGGACC TGAGCCGGTT GGGTCGAGAC CTGCGGCGGG TGCTCATCCT GGACAATTCA 420
CCTGCCTCCT ATGTCTTCCA TCCAGACAAT GCTGTACCGG TGGCCTCGTG GTTTGACAAC 480
ATGAGTGACA CAGAGCTCCA CGACCTCCTC CCCTTCTTCG AGCAACTCAG CCGTGTGGAC 540
GACGTGTACT CAGTGCTCAG GCAGCCACGG CCAGGGAGCT AGTGAGGGTG ATGGGGCCAG 600
GACCTGCCCC TGACCAATGA TACCCACACC TCCTCCCAGG AAGACTGCCC AGGCCTTTGT 660
TAGGAAAACC CATGGGCCGC CGCCACACTC AGTGCCATGG GGAAGCGGGC GTCTCCCCCA 720
CCAGCCCCAC CAGGCGGTGT AGGGGCAGCA GGCTGCACTG AGGACCGTGA GCTCCAGGCC 780
CCGTGTCAGT GCCTTCAAAC CTCCTCCCCT ATTCTCAGGG GACCTGGGGG GCCCTGCCTG 840
CTGCTCCCTT TTTCTGTCTC TGTCCATGCT GCCATGTTTC TCTGCTGCCA AATTGGGCCC 900
CTTGGCCCCT TCCGGTTCTG CTTCCTGGGG GCAGGGTTCC TGCCTTGGAC CCCCAGTCTG 960
GGAACGGTGG ACATCAAGTG CCTTGCATAG AGCCCCCTCT TCCCCGCCCA GCTTTCCCAG 1020
GGGCACAGCT CTAGGCTGGG AGGGGAGAAC CAGCCCCTCC CCCTGCCCCA CCTCCTCCCT 1080
TGGGACTGAG AGGGCCCCTA CCAACCTTTG CCTCTGCCTT GGAGGGAGGG GAGGTCTGTT 1140
ACCACTGGGG AAGGCAGCAG GAGTCTGTCC TTCAGGCCCC ACAGTGCAGC TTCTCCAGGG 1200
CCGACAGCTG AGGGCTGCTC CCTGCATCAT CCAAGCAATG ACCTCAGACT TCTGCCTTAA 1260
CCAGCCCCGG GGCTTGGCTC CCCCAGCTCT GAGCGTGGGG GCATAGGCAG GACCCCCCTT 1320
GTGGTGCCAT ATAAATATGT ACATGTGTAT ATAGATTTTT AGGGGAAGGA GAGAGGGAAG 1380
GGTCAGGGTA GAGACACCCC TCCCTTGCCC CTTTCCTGGG CCCAGAAGTT GGGGGGAGGG 1440
AGGGAAAGGA TTTTTACATT TTTTAAACTG CTATTTTCTG AATGGAACAA GCTGGGCCAA 1500
GGGGCCCAGG CCCTGTCCTC TGTCCCTCAC ACCCCTTTGC TCCGTTCATT CATTCAAAAA 1560
AACATTTCTT GAGCACCTTC TGTGCCCAGC ATATGCTAGG CCCACCAGCT AAGTGTGTGT 1620
GGGGGGTCTC TACGCCAGCT CATCAGTGCC TCCTTGCCCA TCCTTCACCG GTGCCTTTGG 1680
GGGATCTGTA GGAGGTGGGA CCTTCTGTGG GGTTTGGGGA TCTCCAGGAA GCCCGACCAA 1740
GCTGTCCCCT TCCCCTGTGC CAACCCATCT CCTACAGCCC CCTGCCTGAT CCCCTGCTGG 1800
CTGGGGGCAG CTCCCAGGAT ATCCTGCCTT CCAACTGTTT CTGAAGCCCC TCCTCCTAAC 1860
ATGGCGATTC CGGAGGTCAA GGCCTTGGGC TCTCCCCAGG GTCTAACGGT TAAGGGGACC 1920
CACATACCAG TGCCAAGGGG GATGTCAAGT GGTGATGTCG TTGTGCTCCC CTCCCCCAGA 1980
GCGGGTGGGC GGGGGGTGAA TATGGTTGGC CTGCATCAGG TGGCCTTCCC ATTTAAGTGC 2040
CTTCTCTGTG ACTGAGAGCC CTAGTGTGAT GAGAACTAAA GAGAAAGCCA GACCCCTAAA 2100
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 2158
B: aminoacid sequence (SEQ ID NO:14) length: 146 amino acid
1 MLPCFSAAKL GPLAPSGSAS WGQGSCLGPP VWERWTSSAL HRAPSSPPSF PRGTALGWEG
61 RTSPSPCPTS SLGTERAPTN LCLCLGGRGG LLPLGKAAGV CPSGPTVQLL QGRQLRAAPC
121 IIQAMTSDFC LNQPRGLAPP ALSVGA
C. Nucleotide and amino acid composite sequence (SEQ ID NO:15)
Clone number: PP4664
Start code: 866 ATG stop coding: 1306 TAG
Protein molecular weight: 14815
1 G CCA TCC CTA AGC AGA CCC CAG TCC AAT ACC TGC TCC CTG AGG CCA 46
47 AGG CCC AGG ACT CAG ACA AGA TCT GCG TGG TCA TCG ACC TGG ACG AGA 94
95 CCC TGG TGC ACA GCT CCT TCA AGC CAG TGA ACA ACG CGG ACT TCA TCA 142
143 TCC CTG TGG AGA TTG ATG GGG TGG TCC ACC AGG TCT ACG TGT TGA AGC 190
191 GTC CTC ATG TGG ATG AGT TCC TGC AGC GAA TGG GCG AGC TCT TTG AAT 238
239 GTG TGC TGT TCA CTG CTA GCC TCG CCA AGT ACG CAG ACC CAG TAG CTG 286
287 ACC TGC TGG ACA AAT GGG GGG CCT TCC GGG CCC GGC TGT TTC GAG AGT 334
335 CCT GCG TCT TCC ACC GGG GGA ACT ACG TGA AGG ACC TGA GCC GGT TGG 382
383 GTC GAG ACC TGC GGC GGG TGC TCA TCC TGG ACA ATT CAC CTG CCT CCT 430
431 ATG TCT TCC ATC CAG ACA ATG CTG TAC CGG TGG CCT CGT GGT TTG ACA 478
479 ACA TGA GTG ACA CAG AGC TCC ACG ACC TCC TCC CCT TCT TCG AGC AAC 526
527 TCA GCC GTG TGG ACG ACG TGT ACT CAG TGC TCA GGC AGC CAC GGC CAG 574
575 GGA GCT AGT GAG GGT GAT GGG GCC AGG ACC TGC CCC TGA CCA ATG ATA 622
623 CCC ACA CCT CCT CCC AGG AAG ACT GCC CAG GCC TTT GTT AGG AAA ACC 670
671 CAT GGG CCG CCG CCA CAC TCA GTG CCA TGG GGA AGC GGG CGT CTC CCC 718
719 CAC CAG CCC CAC CAG GCG GTG TAG GGG CAG CAG GCT GCA CTG AGG ACC 766
767 GTG AGC TCC AGG CCC CGT GTC AGT GCC TTC AAA CCT CCT CCC CTA TTC 814
815 TCA GGG GAC CTG GGG GGC CCT GCC TGC TGC TCC CTT TTT CTG TCT CTG 862
863 TCC ATG CTG CCA TGT TTC TCT GCT GCC AAA TTG GGC CCC TTG GCC CCT 910
1 Met Leu Pro Cys Phe Ser Ala Ala Lys Leu Gly Pro Leu Ala Pro 15
911 TCC GGT TCT GCT TCC TGG GGG CAG GGT TCC TGC CTT GGA CCC CCA GTC 958
16 Ser Gly Ser Ala Ser Trp Gly Gln Gly Ser Cys Leu Gly Pro Pro Val 31
959 TGG GAA CGG TGG ACA TCA AGT GCC TTG CAT AGA GCC CCC TCT TCC CCG 1006
32 Trp Glu Arg Trp Thr Ser Ser Ala Leu His Arg Ala Pro Ser Ser Pro 47
1007 CCC AGC TTT CCC AGG GGC ACA GCT CTA GGC TGG GAG GGG AGA ACC AGC 1054
48 Pro Ser Phe Pro Arg Gly Thr Ala Leu Gly Trp Glu Gly Arg Thr Ser 63
1055 CCC TCC CCC TGC CCC ACC TCC TCC CTT GGG ACT GAG AGG GCC CCT ACC 1102
64 Pro Ser Pro Cys Pro Thr Ser Ser Leu Gly Thr Glu Arg Ala Pro Thr 79
1103 AAC CTT TGC CTC TGC CTT GGA GGG AGG GGA GGT CTG TTA CCA CTG GGG 1150
80 Asn Leu Cys Leu Cys Leu Gly Gly Arg Gly Gly Leu Leu Pro Leu Gly 95
1151 AAG GCA GCA GGA GTC TGT CCT TCA GGC CCC ACA GTG CAG CTT CTC CAG 1198
96 Lys Ala Ala Gly Val Cys Pro Ser Gly Pro Thr Val Gln Leu Leu Gln 111
1199 GGC CGA CAG CTG AGG GCT GCT CCC TGC ATC ATC CAA GCA ATG ACC TCA 1246
112 Gly Arg Gln Leu Arg Ala Ala Pro Cys Ile Ile Gln Ala Met Thr Ser 127
1247 GAC TTC TGC CTT AAC CAG CCC CGG GGC TTG GCT CCC CCA GCT CTG AGC 1294
128 Asp Phe Cys Leu Asn Gln Pro Arg Gly Leu Ala Pro Pro Ala Leu Ser 143
1295 GTG GGG GCA TAG GCA GGA CCC CCC TTG TGG TGC CAT ATA AAT ATG TAC 1342
144 Val Gly Ala *** 147
1343 ATG TGT ATA TAG ATT TTT AGG GGA AGG AGA GAG GGA AGG GTC AGG GTA 1390
1391 GAG ACA CCC CTC CCT TGC CCC TTT CCT GGG CCC AGA AGT TGG GGG GAG 1438
1439 GGA GGG AAA GGA TTT TTA CAT TTT TTA AAC TGC TAT TTT CTG AAT GGA 1486
1487 ACA AGC TGG GCC AAG GGG CCC AGG CCC TGT CCT CTG TCC CTC ACA CCC 1534
1535 CTT TGC TCC GTT CAT TCA TTC AAA AAA ACA TTT CTT GAG CAC CTT CTG 1582
1583 TGC CCA GCA TAT GCT AGG CCC ACC AGC TAA GTG TGT GTG GGG GGT CTC 1630
1631 TAC GCC AGC TCA TCA GTG CCT CCT TGC CCA TCC TTC ACC GGT GCC TTT 1678
1679 GGG GGA TCT GTA GGA GGT GGG ACC TTC TGT GGG GTT TGG GGA TCT CCA 1726
1727 GGA AGC CCG ACC AAG CTG TCC CCT TCC CCT GTG CCA ACC CAT CTC CTA 1774
1775 CAG CCC CCT GCC TGA TCC CCT GCT GGC TGG GGG CAG CTC CCA GGA TAT 1822
1823 CCT GCC TTC CAA CTG TTT CTG AAG CCC CTC CTC CTA ACA TGG CGA TTC 1870
1871 CGG AGG TCA AGG CCT TGG GCT CTC CCC AGG GTC TAA CGG TTA AGG GGA 1918
1919 CCC ACA TAC CAG TGC CAA GGG GGA TGT CAA GTG GTG ATG TCG TTG TGC 1966
1967 TCC CCT CCC CCA GAG CGG GTG GGC GGG GGG TGA ATA TGG TTG GCC TGC 2014
2015 ATC AGG TGG CCT TCC CAT TTA AGT GCC TTC TCT GTG ACT GAG AGC CCT 2062
2063 AGT GTG ATG AGA ACT AAA GAG AAA GCC AGA CCC CTA AAA AAA AAA AAA 2110
2111 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 2158
6.PP4748 albumen
A: nucleotide sequence (SEQ ID NO:16) length: 1337bp
GAACAGCGGG GGCACACGGG GCGCTGCCGA AGTGCAAGGC CACGGCCAGA GCTCGAGCCC 60
GACGCGCTGT CTGGAGTCGT AGGTTGGCGC CGTTTGGGGT CGGGGTCTGA GGCTTGGGCG 120
CTGCCTGGGC CGAGCGGAGA TCGGGGTTTG CCTCCCGTCC CCGCTCAGGA CCCTGACGTG 180
GCTGAAGCGG CCCCGGGAGC ATGAGCGGGC AGCGCGTGGA CGTCAAGGTG GTGATGCTGG 240
GCAAGGAGTA CGTGGGCAAG ACTAGCCTGG TGGAGCGCTA CGTGCACGAC CGCTTTCTGG 300
TGGGGCCTTA TCAGAACACC ATCGGGGCCG CCTTCGTGGC CAAGGTGATG TCGGTCGGAG 360
ACCGGACTGT GACATTAGGT ATTTGGGACA CAGCAGGCTC TGAGCGCTAT GAGGCCATGA 420
GTAGAATCTA CTATCGGGGT GCCAAGGCTG CCATCGTCTG CTATGACCTC ACAGACAGCA 480
GCAGCTTTGA GCGAGCAAAG TTCTGGGTGA AGGAACTGCG CAGCCTAGAG GAGGGCTGCC 540
AAATCTACTT ATGTGGCACC AAGAGTGACC TGCTGGAAGA AGACCGGAGG CGTCGACGTG 600
TGGACTTCCA CGACGTCCAG GACTATGCAG ACAGTAGCTG CTCCTCAGCC CTTTGGGGGG 660
TGGGGGTGTG TGGCTGTCTG GGTGGATCAA AGAAAATAGG GACTGCCTTG GCTGCCAGGG 720
CAAGGTGCTC TAGGAGGTCT TCCTGGCCTC CTTGAACTGT GGGGTCCAGG AGACTCCCTG 780
AACTGCTAGC CCTCCCTTTT GTCTGTTTAT CTAATTCTCA GGTATGAGGC TTTAGTCACT 840
TCTCTTTACA GATATCAAAG CTCAGCTCTT TGAAACATCC AGCAAGACAG GCCAGAGTGT 900
GGACGAGCTC TTCCAGAAAG TGGCAGAGGA TTACGTCAGT GTGGCTGCCT TCCAGGTGAT 960
GACAGAGGAC AAGGGCGTGG ATCTGGGCCA GAAGCCAAAC CCCTACTTCT ACAGCTGTTG 1020
TCATCACTGA GTCAGCACTC ACCTGGCCTG GGGGAATTAA AGGAATTCCC CGTAAGGGCT 1080
GGACCCAGCT CCTTTCTGGG CTTGGGTAGT CAAATGTCTG AGCTACCCCA GGTCCTCATG 1140
TCAGCAGAGT GGCGCCTGCC TGTGCTGGCC CATGGAACGG AGACAGCATT GGGCTGACTG 1200
TGGGCATGAG GAGGGATAAG GCTGATTTGG ACCCCAGGCT TCTGCCCTGG ACAGCACTTG 1260
TGTCTGCAGA TTATTTAAGT GGCTTTTGAT CTGTAAATAA AATCAGTGCA CTGTGCATCA 1320
AAAAAAAAAA AAAAAAA 1337
B: aminoacid sequence (SEQ ID NO:17) length: 184 amino acid
1 MSGQRVDVKV VMLGKEYVGK TSLVERYVHD RFLVGPYQNT IGAAFVAKVM SVGDRTVTLG
61 IWDTAGSERY EAMSRIYYRG AKAAIVCYDL TDSSSFERAK FWVKELRSLE EGCQIYLCGT
121 KSDLLEEDRR RRRVDFHDVQ DYADSSCSSA LWGVGVCGCL GGSKKIGTAL AARARCSRRS
181 SWPP
C. Nucleotide and amino acid composite sequence (SEQ ID NO:18)
Clone number: PP4748
Start code: 201 ATG stop coding: 755 TGA
Protein molecular weight: 20517
1 GA ACA GCG GGG GCA CAC GGG GCG CTG CCG AAG TGC AAG GCC ACG GCC 47
48 AGA GCT CGA GCC CGA CGC GCT GTC TGG AGT CGT AGG TTG GCG CCG TTT 95
96 GGG GTC GGG GTC TGA GGC TTG GGC GCT GCC TGG GCC GAG CGG AGA TCG 143
144 GGG TTT GCC TCC CGT CCC CGC TCA GGA CCC TGA CGT GGC TGA AGC GGC 191
192 CCC GGG AGC ATG AGC GGG CAG CGC GTG GAC GTC AAG GTG GTG ATG CTG 239
1 Met Ser Gly Gln Arg Val Asp Val Lys Val Val Met Leu 13
240 GGC AAG GAG TAC GTG GGC AAG ACT AGC CTG GTG GAG CGC TAC GTG CAC 287
14 Gly Lys Glu Tyr Val Gly Lys Thr Ser Leu Val Glu Arg Tyr Val His 29
288 GAC CGC TTT CTG GTG GGG CCT TAT CAG AAC ACC ATC GGG GCC GCC TTC 335
30 Asp Arg Phe Leu Val Gly Pro Tyr Gln Asn Thr Ile Gly Ala Ala Phe 45
336 GTG GCC AAG GTG ATG TCG GTC GGA GAC CGG ACT GTG ACA TTA GGT ATT 383
46 Val Ala Lys Val Met Ser Val Gly Asp Arg Thr Val Thr Leu Gly Ile 61
384 TGG GAC ACA GCA GGC TCT GAG CGC TAT GAG GCC ATG AGT AGA ATC TAC 431
62 Trp Asp Thr Ala Gly Ser Glu Arg Tyr Glu Ala Met Ser Arg Ile Tyr 77
432 TAT CGG GGT GCC AAG GCT GCC ATC GTC TGC TAT GAC CTC ACA GAC AGC 479
78 Tyr Arg Gly Ala Lys Ala Ala Ile Val Cys Tyr Asp Leu Thr Asp Ser 93
480 AGC AGC TTT GAG CGA GCA AAG TTC TGG GTG AAG GAA CTG CGC AGC CTA 527
94 Ser Ser Phe Glu Arg Ala Lys Phe Trp Val Lys Glu Leu Arg Ser Leu 109
528 GAG GAG GGC TGC CAA ATC TAC TTA TGT GGC ACC AAG AGT GAC CTG CTG 575
110 Glu Glu Gly Cys Gln Ile Tyr Leu Cys Gly Thr Lys Ser Asp Leu Leu 125
576 GAA GAA GAC CGG AGG CGT CGA CGT GTG GAC TTC CAC GAC GTC CAG GAC 623
126 Glu Glu Asp Arg Arg Arg Arg Arg Val Asp Phe His Asp Val Gln Asp 141
624 TAT GCA GAC AGT AGC TGC TCC TCA GCC CTT TGG GGG GTG GGG GTG TGT 671
142 Tyr Ala Asp Ser Ser Cys Ser Ser Ala Leu Trp Gly Val Gly Val Cys 157
672 GGC TGT CTG GGT GGA TCA AAG AAA ATA GGG ACT GCC TTG GCT GCC AGG 719
158 Gly Cys Leu Gly Gly Ser Lys Lys Ile Gly Thr Ala Leu Ala Ala Arg 173
720 GCA AGG TGC TCT AGG AGG TCT TCC TGG CCT CCT TGA ACT GTG GGG TCC 767
174 Ala Arg Cys Ser Arg Arg Ser Ser Trp Pro Pro *** 185
768 AGG AGA CTC CCT GAA CTG CTA GCC CTC CCT TTT GTC TGT TTA TCT AAT 815
816 TCT CAG GTA TGA GGC TTT AGT CAC TTC TCT TTA CAG ATA TCA AAG CTC 863
864 AGC TCT TTG AAA CAT CCA GCA AGA CAG GCC AGA GTG TGG ACG AGC TCT 911
912 TCC AGA AAG TGG CAG AGG ATT ACG TCA GTG TGG CTG CCT TCC AGG TGA 959
960 TGA CAG AGG ACA AGG GCG TGG ATC TGG GCC AGA AGC CAA ACC CCT ACT 1007
1008 TCT ACA GCT GTT GTC ATC ACT GAG TCA GCA CTC ACC TGG CCT GGG GGA 1055
1056 ATT AAA GGA ATT CCC CGT AAG GGC TGG ACC CAG CTC CTT TCT GGG CTT 1103
1104 GGG TAG TCA AAT GTC TGA GCT ACC CCA GGT CCT CAT GTC AGC AGA GTG 1151
1152 GCG CCT GCC TGT GCT GGC CCA TGG AAC GGA GAC AGC ATT GGG CTG ACT 1199
1200 GTG GGC ATG AGG AGG GAT AAG GCT GAT TTG GAC CCC AGG CTT CTG CCC 1247
1248 TGG ACA GCA CTT GTG TCT GCA GAT TAT TTA AGT GGC TTT TGA TCT GTA 1295
1296 AAT AAA ATC AGT GCA CTG TGC ATC AAA AAA AAA AAA AAA AAA 1337
D.Blastp result
Query=PP4748 albumen (184 amino acid)
>SP_IN：P91857 P91857 caenorhabditis elegans.f26h9.6 protein.11/1999
Length=208 amino acid
Score value=108bits (266), predicated value=3e-23
Homogeny=60/140 (42%), similarity=88/140 (62%), breach=8/140 (5%)
Query：9 KVVMLGKEYVGKTSLVERYVHDRFLVGPYQ-NTIGAAFVAKVMSVGDRTVTLGIWDTAGS 67
K+V+LG+ VGK+SLV R+V +F YQ +TIGAAF+ + + + D T+ IWDTAG
Sbjct：21 KLVLLGESAVGKSSLVLRFVKGQF--HEYQESTIGAAFLTQTVCLDDATIKFEIWDTAGQ 78
Query：68 ERYEAMSRIYYRGAKAAIVCYDLTDSSSFERAKFWVKEL-RSLEEGCQIYLCGTKSXXXX 126
ERY +++ +YYRGA+AAIV YD+T+ SF++AK WVKEL R +L G K+
Sbjct：79 ERYHSLAPMYYRGAQAAIVVYDITNQESFQKAKNWVKELQRQASPNIVMALAGNKA---- 134
Query：127 XXXXXXXVDFHDVQDYADSS 146
V++ + YA+ +
Sbjct：135 DYANKRTVEYEEANAYAEDN 154
7.PP4762 albumen
A: nucleotide sequence (SEQ ID NO:19) length: 2417bp
GTTTCTGGCG CGAACTTCCG CCGTTCCGAA GTTGCACGGT GAATTGGCGC TATGTCTGGG 60
GACAGCAGCG GCCGCGGGCC AGAGGGCCGG GGCCGGGGCC GCGACCCGCA TCGGGATCGC 120
ACCCGCTCCC GCTCCCGCTC GCGGTCCCCT TTGTCGCCCA GGTCCCGCCG CGGCTCTGCG 180
CGGGAGCGCA GAGAGGCCCC AGAGCGCCCG AGCCTGGAGG ACACAGAGCC GTCGGATTCC 240
GGGGACGAGA TGATGGACCC GGCCAGCTTG GAGGCGGAGG CCGACCAAGG CCTGTGCCGC 300
CAGATCCGCC ATCAGTACCG GGCGCTCATC AACTCCGTCC AACGTAAGGC GGCGCCTCCG 360
GGCGGCGCGG GCCCGGACGG GCCGCTGTCC CCGCCCTGCG CCGGGCGCGG GGGGAGGGCA 420
CCGGAGGCAG GTGCCGGGCG GAGGCCCGGC GCGGCGTTAG TAGAGACGCG CCGGGTGCCA 480
GCCCCTCGCC GCACCTTAAA TTGTTGACAA AGTGCTCCCA CTTTGTGGCT GGAGCCGTAC 540
GACCTTGGCG CGGGTCCCCG AGTGGCGGGC TGGAAAAGCT GGAGGCCAGA GCCGTTCGCG 600
GACTCGCCCG AGCCCAGCCA GCCAGTGGGG AGAGGCAGGA CGCGGACTCC GCGTGCTTTG 660
ATTGCGCCCG GCAGTCCTCG GCGTGGGCTC CCGGCCTCGC AGCGGCACGC TGAGCTGCAG 720
CGGCACGCGA GAGCTGTCAA ATGCACGTTC TCGGACCCCA CCCGGACCCG CGGAATTCGA 780
ATCTCTGGGT GGGTCCGCGC AACTGTGGAG TGGCCCTCCA GGTGATGGTG TGTATTGCTT 840
GCAAAGTTTG AGAACCACTC AGTTATGGAG CACTCAGAGG CTGCAGCCGG AGTCCAGCCC 900
TTTGGGTTAA GGTTAGAGGG AGCTAGGGTT TCCTTTTAAA ATTCATGCTG CATAAAGGGT 960
AGGGTGGCCT TTCTCCTTTT CCGGGTCATT TTGGTTAAAA AGATAAAGAT GTTCGAAGTA 1020
ATTACTCAGA GCGCAGGGGC CGGGGCTTGG CTTTACTGTT GCAGGTGATA ACCTTGTCAA 1080
CTGCTTATTA GTGAATGCTG AACAAATTTC CCAAAGTTTG TATTTTTAAA AATAGAAAAC 1140
CGTGAGACAT ACTGAATGCC GGTGACAAAT TAACAGAGGT CCTTGAAGAG GCTAACACTC 1200
TGTTTAATGA AGTGTCCCGA GCAAGAGAAG CAGTCCTGGA TGCCCACTTT CTTGTTTTGG 1260
CTTCAGATTT GGGCAAAGAG AAAGCAAAGC AGCTGCGCTC AGACCTGAGC TCCTTTGACA 1320
TGTTAAGATA TGTTGAAACT CTACTCACAC ATATGGGTGT AAATCCGCTA GAAGCTGAAG 1380
AACTCATCCG TGATGAAGAT AGTCCTGATT TTGAATTCAT AGTCTATGAC TCCTGGAAGA 1440
TAACAGGCAG AACAGCAGAA AACACCTTTA ATAAAACCCA TACATTCCAC TTTCTGTTGG 1500
GTTCAATATA CGGAGAGTGC CCTGTGCCAA AGCCACGAGT TGATCGTCCA AGAAAAGTTC 1560
CTGTGATACA AGAGGAGAGG GCAATGCCTG CCCAGTTAAG AAGAATGGAA GAATCTCATC 1620
AAGAAGCAAC AGAGAAAGAA GTAGAAAGAA TCTTGGGATT GTTGCAGACA TATTTTCGAG 1680
AAGATCGAGA CAGTCTCACT CTTTCGCCCA GACTGGAATG CAGTGGCACC ATCTCAACTC 1740
ACTGCAACCT CCACCTCCTG GATTCAAGCA ATTCTCCTGC CTCAGCCTCC TGAGTAGCTG 1800
GGATTACAGG CGCACGCGCC ACCACATCTG GCTAATTTTT TTTCTGTATT TTTAGTAGAG 1860
ACATTGTTTC GCCATGTTGG CCAAGCTAGT CTTGAACCCC TGACCTCAGG TGATCTGCCA 1920
GCATCGGCCT CCCAAAGTGC TGGGATTACA GGTCTGATAC CCCAATGTCC TTCTTTGACT 1980
TTGTGGTTGA TCCTCATTCT TTCCCCCGTA CAGTGGAAAA CATCTTTCAT GTTTCCTTCA 2040
TTATACGGGA TGGTTTTGCA AGAATAAGAC TTGACCAAGA CCGACTGCCA GTAATAGAGC 2100
CTGTTAGTAT TAATGAAGAA AATGAGGGAT TTGAACATAA CACACAAGTT AGAAATCAAG 2160
GAATTATAGC TTTGAGTTAC CGTGACTGGG AGGAGATTGT GAAGACCTTT GAGATTTCAG 2220
AGCCTGTGAT TACTCCAAGT CAGAGGCAGC AGAAGCCAAG TGCTTGATGC TAGCTGAAGG 2280
ACTCAAATGG ATAGTGAAGT CCAAAACGGA AAGCGGCATG TATTGTACAT ATTGTATGAT 2340
TCAACATTTT TAAAGGCAGA TTGTTTTTAG TAAAATGTAG CTTTTGATAG TTAAAAAAAA 2400
AAAAAAAAAA AAAAAAA 2417
B: aminoacid sequence (SEQ ID NO:20) length: 157 amino acid
1 MLRYVETLLT HMGVNPLEAE ELIRDEDSPD FEFIVYDSWK ITGRTAENTF NKTHTFHFLL
61 GSIYGECPVP KPRVDRPRKV PVIQEERAMP AQLRRMEESH QEATEKEVER ILGLLQTYFR
121 EDRDSLTLSP RLECSGTIST HCNLHLLDSS NSPASAS
C. Nucleotide and amino acid composite sequence (SEQ ID NO:21)
Clone number: PP4762
Start code: 1320 ATG stop coding: 1793 TGA
Protein molecular weight: 18060
1 GT TTC TGG CGC GAA CTT CCG CCG TTC CGA AGT TGC ACG GTG AAT TGG 47
48 CGC TAT GTC TGG GGA CAG CAG CGG CCG CGG GCC AGA GGG CCG GGG CCG 95
96 GGG CCG CGA CCC GCA TCG GGA TCG CAC CCG CTC CCG CTC CCG CTC GCG 143
144 GTC CCC TTT GTC GCC CAG GTC CCG CCG CGG CTC TGC GCG GGA GCG CAG 191
192 AGA GGC CCC AGA GCG CCC GAG CCT GGA GGA CAC AGA GCC GTC GGA TTC 239
240 CGG GGA CGA GAT GAT GGA CCC GGC CAG CTT GGA GGC GGA GGC CGA CCA 287
288 AGG CCT GTG CCG CCA GAT CCG CCA TCA GTA CCG GGC GCT CAT CAA CTC 335
336 CGT CCA ACG TAA GGC GGC GCC TCC GGG CGG CGC GGG CCC GGA CGG GCC 383
384 GCT GTC CCC GCC CTG CGC CGG GCG CGG GGG GAG GGC ACC GGA GGC AGG 431
432 TGC CGG GCG GAG GCC CGG CGC GGC GTT AGT AGA GAC GCG CCG GGT GCC 479
480 AGC CCC TCG CCG CAC CTT AAA TTG TTG ACA AAG TGC TCC CAC TTT GTG 527
528 GCT GGA GCC GTA CGA CCT TGG CGC GGG TCC CCG AGT GGC GGG CTG GAA 575
576 AAG CTG GAG GCC AGA GCC GTT CGC GGA CTC GCC CGA GCC CAG CCA GCC 623
624 AGT GGG GAG AGG CAG GAC GCG GAC TCC GCG TGC TTT GAT TGC GCC CGG 671
672 CAG TCC TCG GCG TGG GCT CCC GGC CTC GCA GCG GCA CGC TGA GCT GCA 719
720 GCG GCA CGC GAG AGC TGT CAA ATG CAC GTT CTC GGA CCC CAC CCG GAC 767
768 CCG CGG AAT TCG AAT CTC TGG GTG GGT CCG CGC AAC TGT GGA GTG GCC 815
816 CTC CAG GTG ATG GTG TGT ATT GCT TGC AAA GTT TGA GAA CCA CTC AGT 863
864 TAT GGA GCA CTC AGA GGC TGC AGC CGG AGT CCA GCC CTT TGG GTT AAG 911
912 GTT AGA GGG AGC TAG GGT TTC CTT TTA AAA TTC ATG CTG CAT AAA GGG 959
960 TAG GGT GGC CTT TCT CCT TTT CCG GGT CAT TTT GGT TAA AAA GAT AAA 1007
1008 GAT GTT CGA AGT AAT TAC TCA GAG CGC AGG GGC CGG GGC TTG GCT TTA 1055
1056 CTG TTG CAG GTG ATA ACC TTG TCA ACT GCT TAT TAG TGA ATG CTG AAC 1103
1104 AAA TTT CCC AAA GTT TGT ATT TTT AAA AAT AGA AAA CCG TGA GAC ATA 1151
1152 CTG AAT GCC GGT GAC AAA TTA ACA GAG GTC CTT GAA GAG GCT AAC ACT 1199
1200 CTG TTT AAT GAA GTG TCC CGA GCA AGA GAA GCA GTC CTG GAT GCC CAC 1247
1248 TTT CTT GTT TTG GCT TCA GAT TTG GGC AAA GAG AAA GCA AAG CAG CTG 1295
1296 CGC TCA GAC CTG AGC TCC TTT GAC ATG TTA AGA TAT GTT GAA ACT CTA 1343
1 Met Leu Arg Tyr Val Glu Thr Leu 8
1344 CTC ACA CAT ATG GGT GTA AAT CCG CTA GAA GCT GAA GAA CTC ATC CGT 1391
9 Leu Thr His Met Gly Val Asn Pro Leu Glu Ala Glu Glu Leu Ile Arg 24
1392 GAT GAA GAT AGT CCT GAT TTT GAA TTC ATA GTC TAT GAC TCC TGG AAG 1439
25 Asp Glu Asp Ser Pro Asp Phe Glu Phe Ile Val Tyr Asp Ser Trp Lys 40
1440 ATA ACA GGC AGA ACA GCA GAA AAC ACC TTT AAT AAA ACC CAT ACA TTC 1487
41 Ile Thr Gly Arg Thr Ala Glu Asn Thr Phe Asn Lys Thr His Thr Phe 56
1488 CAC TTT CTG TTG GGT TCA ATA TAC GGA GAG TGC CCT GTG CCA AAG CCA 1535
57 His Phe Leu Leu Gly Ser Ile Tyr Gly Glu Cys Pro Val Pro Lys Pro 72
1536 CGA GTT GAT CGT CCA AGA AAA GTT CCT GTG ATA CAA GAG GAG AGG GCA 1583
73 Arg Val Asp Arg Pro Arg Lys Val Pro Val Ile Gln Glu Glu Arg Ala 88
1584 ATG CCT GCC CAG TTA AGA AGA ATG GAA GAA TCT CAT CAA GAA GCA ACA 1631
89 Met Pro Ala Gln Leu Arg Arg Met Glu Glu Ser His Gln Glu Ala Thr 104
1632 GAG AAA GAA GTA GAA AGA ATC TTG GGA TTG TTG CAG ACA TAT TTT CGA 1679
105 Glu Lys Glu Val Glu Arg Ile Leu Gly Leu Leu Gln Thr Tyr Phe Arg 120
1680 GAA GAT CGA GAC AGT CTC ACT CTT TCG CCC AGA CTG GAA TGC AGT GGC 1727
121 Glu Asp Arg Asp Ser Leu Thr Leu Ser Pro Arg Leu Glu Cys Ser Gly 136
1728 ACC ATC TCA ACT CAC TGC AAC CTC CAC CTC CTG GAT TCA AGC AAT TCT 1775
137 Thr Ile Ser Thr His Cys Asn Leu His Leu Leu Asp Ser Ser Asn Ser 152
1776 CCT GCC TCA GCC TCC TGA GTA GCT GGG ATT ACA GGC GCA CGC GCC ACC 1823
153 Pro Ala Ser Ala Ser *** 158
1824 ACA TCT GGC TAA TTT TTT TTC TGT ATT TTT AGT AGA GAC ATT GTT TCG 1871
1872 CCA TGT TGG CCA AGC TAG TCT TGA ACC CCT GAC CTC AGG TGA TCT GCC 1919
1920 AGC ATC GGC CTC CCA AAG TGC TGG GAT TAC AGG TCT GAT ACC CCA ATG 1967
1968 TCC TTC TTT GAC TTT GTG GTT GAT CCT CAT TCT TTC CCC CGT ACA GTG 2015
2016 GAA AAC ATC TTT CAT GTT TCC TTC ATT ATA CGG GAT GGT TTT GCA AGA 2063
2064 ATA AGA CTT GAC CAA GAC CGA CTG CCA GTA ATA GAG CCT GTT AGT ATT 2111
2112 AAT GAA GAA AAT GAG GGA TTT GAA CAT AAC ACA CAA GTT AGA AAT CAA 2159
2160 GGA ATT ATA GCT TTG AGT TAC CGT GAC TGG GAG GAG ATT GTG AAG ACC 2207
2208 TTT GAG ATT TCA GAG CCT GTG ATT ACT CCA AGT CAG AGG CAG CAG AAG 2255
2256 CCA AGT GCT TGA TGC TAG CTG AAG GAC TCA AAT GGA TAG TGA AGT CCA 2303
2304 AAA CGG AAA GCG GCA TGT ATT GTA CAT ATT GTA TGA TTC AAC ATT TTT 2351
2352 AAA GGC AGA TTG TTT TTA GTA AAA TGT AGC TTT TGA TAG TTA AAA AAA 2399
2400 AAA AAA AAA AAA AAA AAA 2417
8.PP5242 albumen
A: nucleotide sequence (SEQ ID NO:22) length: 1685bp
GCACCGCGGA GGACAGGGGC AGCTGGCGGG CAGCGGGTGA GGGGGTGGCG GGGACGCGAG 60
TGGCGGCCGC GGGGCCCCGG ACAAGGGTCC GCAGAGCTGC AGCCTTCGAG GGCCAGCCCT 120
CTCCGAGTCC GGGGCTGGGT CCCACCAGTG ACAAGGCGGC AGCCCCGCGC ACACCAAAGA 180
GAAGGCGGCT GTGGCGGCAG CGGCAGCCCC AGCCATGCTG TGTTATGTGA CGAGGCCGGA 240
CGCGGTGCTG ATGGAGGTGG AGGTGGAGGC GAAAGCCAAC GGCGAGGACT GCCTCAACCA 300
GGTGTGCAGG CGACTGGGAA TCATAGAAGT TGACTATTTT GGACTGCAGT TTACGGGTAG 360
CAAAGGTGAA AGTTTATGGC TAAACCTGAG AAACCGGATC TCCCAGCAGA TGGATGGGCT 420
AGCCCCTTAC AGGCTTAAAC TTAGAGTCAA GTTCTTCGTG GAGCCTCATC TCATCTTACA 480
GGAGCAGACT AGGCATATCT TTTTCTTGCA CATCAAGGAG GCCCTCTTGG CAGGCCACCT 540
CTTGTGTTCC CCAGAGCAGG CAGTGGAACT CAGTGCCCTC CTGGCCCAGA CCAAGTTTGG 600
AGACTACAAC CAGAACACTG CCAAGTATAA CTATGAGGAG CTCTGTGCCA AGGAGCTCTC 660
CTCTGCCACC TTGAACAGCA TTGTTGCAAA ACATAAGGAG TTGGAGGGGA CCAGCCAGGC 720
TTCAGCTGAA TACCAAGTTT TGCAGATTGT GTCGGCAATG GAAAACTATG GCATAGAATG 780
GCATTCTGTG CGGGATAGCG AAGGGCAGAA ACTGCTCATT GGGGTTGGAC CTGAAGGAAT 840
CTCAATTTGT AAAGATGACT TTAGCCCAAT TAATAGGATA GCTTATCCTG TGGTGCAGAT 900
GGCCACCCAG TCAGGAAAGA ATGTATATTT GACGGTCACC AAGGAATCTG GGAACAGCAT 960
CGTGCTCTTG TTTAAAATGA TCAGCACCAG GGCGGCCAGC GGGCTCTACC GAGCGATAAC 1020
AGAGACGCAC GCATTCTACA GGTGTGACAC AGTGACCAGC GCCGTGATGA TGCAGTATAG 1080
CCGTGACTTG AAGGGCCACT TGGCATCTCT GTTTCTGAAT GAAAACATTA ACCTTGGCAA 1140
GAAATATGTC TTTGATATTA AAAGAACATC AAAGGAGGTG TATGACCATG CCAGGAGGGC 1200
TCTGTACAAT GCTGGCGTTG TGGACCTCGT TTCAAGAAGC AACCAGAGCC CTTCACACTC 1260
GCCTCTGAAG TCCTCAGAAA GCAGCATGAA CTGCAGCAGC TGCGAGGGCC TCAGCTGCCA 1320
GCAGACCCGG GTGCTGCAGG AGAAGCTACG CAAGCTGAAG GAAGCCATGC TGTGCATGGT 1380
GTGCTGCGAG GAGGAGATCA ACTCCACCTT CTGTCCCTGT GGCCACACTG TGTGCTGTGA 1440
GAGCTGCGCC GCCCAGCTAC AGTCATGTCC CGTCTGCAGG TCGCGTGTGG AGCATGTCCA 1500
GCACGTCTAT CTGCCAACGC ACACCAGTCT TCTCAATCTG ACTGTAATCT AATCTGTTGT 1560
GCTTTTGTTG GACTTGGCAT GTTTCCATGA ACTGCACTAT TATAAACTAT TAAAATGATA 1620
GATTGTGGAG AAAGTAATTA TTCCAACACC CATCTGCCAT GCGATGTTAA AAAAAAAAAA 1680
AAAAA 1685
B: aminoacid sequence (SEQ ID NO:23) length: 445 amino acid
1 MLCYVTRPDA VLMEVEVEAK ANGEDCLNQV CRRLGIIEVD YFGLQFTGSK GESLWLNLRN
61 RISQQMDGLA PYRLKLRVKF FVEPHLILQE QTRHIFFLHI KEALLAGHLL CSPEQAVELS
121 ALLAQTKFGD YNQNTAKYNY EELCAKELSS ATLNSIVAKH KELEGTSQAS AEYQVLQIVS
181 AMENYGIEWH SVRDSEGQKL LIGVGPEGIS ICKDDFSPIN RIAYPVVQMA TQSGKNVYLT
241 VTKESGNSIV LLFKMISTRA ASGLYRAITE THAFYRCDTV TSAVMMQYSR DLKGHLASLF
301 LNENINLGKK YVFDIKRTSK EVYDHARRAL YNAGVVDLVS RSNQSPSHSP LKSSESSMNC
361 SSCEGLSCQQ TRVLQEKLRK LKEAMLCMVC CEEEINSTFC PCGHTVCCES CAAQLQSCPV
421 CRSRVEHVQH VYLPTHTSLL NLTVI
C. Nucleotide and amino acid composite sequence (SEQ ID NO:24)
Clone number: PP5242
Start code: 215 ATG stop coding: 1552 TAA
Protein molecular weight: 49881
1 G CAC CGC GGA GGA CAG GGG CAG CTG GCG GGC AGC GGG TGA GGG GGT 46
47 GGC GGG GAC GCG AGT GGC GGC CGC GGG GCC CCG GAC AAG GGT CCG CAG 94
95 AGC TGC AGC CTT CGA GGG CCA GCC CTC TCC GAG TCC GGG GCT GGG TCC 142
143 CAC CAG TGA CAA GGC GGC AGC CCC GCG CAC ACC AAA GAG AAG GCG GCT 190
191 GTG GCG GCA GCG GCA GCC CCA GCC ATG CTG TGT TAT GTG ACG AGG CCG 238
1 Met Leu Cys Tyr Val Thr Arg Pro 8
239 GAC GCG GTG CTG ATG GAG GTG GAG GTG GAG GCG AAA GCC AAC GGC GAG 286
9 Asp Ala Val Leu Met Glu Val Glu Val Glu Ala Lys Ala Asn Gly Glu 24
287 GAC TGC CTC AAC CAG GTG TGC AGG CGA CTG GGA ATC ATA GAA GTT GAC 334
25 Asp Cys Leu Asn Gln Val Cys Arg Arg Leu Gly Ile Ile Glu Val Asp 40
335 TAT TTT GGA CTG CAG TTT ACG GGT AGC AAA GGT GAA AGT TTA TGG CTA 382
41 Tyr Phe Gly Leu Gln Phe Thr Gly Ser Lys Gly Glu Ser Leu Trp Leu 56
383 AAC CTG AGA AAC CGG ATC TCC CAG CAG ATG GAT GGG CTA GCC CCT TAC 430
57 Asn Leu Arg Asn Arg Ile Ser Gln Gln Met Asp Gly Leu Ala Pro Tyr 72
431 AGG CTT AAA CTT AGA GTC AAG TTC TTC GTG GAG CCT CAT CTC ATC TTA 478
73 Arg Leu Lys Leu Arg Val Lys Phe Phe Val Glu Pro His Leu Ile Leu 88
479 CAG GAG CAG ACT AGG CAT ATC TTT TTC TTG CAC ATC AAG GAG GCC CTC 526
89 Gln Glu Gln Thr Arg His Ile Phe Phe Leu His Ile Lys Glu Ala Leu 104
527 TTG GCA GGC CAC CTC TTG TGT TCC CCA GAG CAG GCA GTG GAA CTC AGT 574
105 Leu Ala Gly His Leu Leu Cys Ser Pro Glu Gln Ala Val Glu Leu Ser 120
575 GCC CTC CTG GCC CAG ACC AAG TTT GGA GAC TAC AAC CAG AAC ACT GCC 622
121 Ala Leu Leu Ala Gln Thr Lys Phe Gly Asp Tyr Asn Gln Asn Thr Ala 136
623 AAG TAT AAC TAT GAG GAG CTC TGT GCC AAG GAG CTC TCC TCT GCC ACC 670
137 Lys Tyr Asn Tyr Glu Glu Leu Cys Ala Lys Glu Leu Ser Ser Ala Thr 152
671 TTG AAC AGC ATT GTT GCA AAA CAT AAG GAG TTG GAG GGG ACC AGC CAG 718
153 Leu Asn Ser Ile Val Ala Lys His Lys Glu Leu Glu Gly Thr Ser Gln 168
719 GCT TCA GCT GAA TAC CAA GTT TTG CAG ATT GTG TCG GCA ATG GAA AAC 766
169 Ala Ser Ala Glu Tyr Gln Val Leu Gln Ile Val Ser Ala Met Glu Asn 184
767 TAT GGC ATA GAA TGG CAT TCT GTG CGG GAT AGC GAA GGG CAG AAA CTG 814
185 Tyr Gly Ile Glu Trp His Ser Val Arg Asp Ser Glu Gly Gln Lys Leu 200
815 CTC ATT GGG GTT GGA CCT GAA GGA ATC TCA ATT TGT AAA GAT GAC TTT 862
201 Leu Ile Gly Val Gly Pro Glu Gly Ile Ser Ile Cys Lys Asp Asp Phe 216
863 AGC CCA ATT AAT AGG ATA GCT TAT CCT GTG GTG CAG ATG GCC ACC CAG 910
217 Ser Pro Ile Asn Arg Ile Ala Tyr Pro Val Val Gln Met Ala Thr Gln 232
911 TCA GGA AAG AAT GTA TAT TTG ACG GTC ACC AAG GAA TCT GGG AAC AGC 958
233 Ser Gly Lys Asn Val Tyr Leu Thr Val Thr Lys Glu Ser Gly Asn Ser 248
959 ATC GTG CTC TTG TTT AAA ATG ATC AGC ACC AGG GCG GCC AGC GGG CTC 1006
249 Ile Val Leu Leu Phe Lys Met Ile Ser Thr Arg Ala Ala Ser Gly Leu 264
1007 TAC CGA GCG ATA ACA GAG ACG CAC GCA TTC TAC AGG TGT GAC ACA GTG 1054
265 Tyr Arg Ala Ile Thr Glu Thr His Ala Phe Tyr Arg Cys Asp Thr Val 280
1055 ACC AGC GCC GTG ATG ATG CAG TAT AGC CGT GAC TTG AAG GGC CAC TTG 1102
281 Thr Ser Ala Val Met Met Gln Tyr Ser Arg Asp Leu Lys Gly His Leu 296
1103 GCA TCT CTG TTT CTG AAT GAA AAC ATT AAC CTT GGC AAG AAA TAT GTC 1150
297 Ala Ser Leu Phe Leu Asn Glu Asn Ile Asn Leu Gly Lys Lys Tyr Val 312
1151 TTT GAT ATT AAA AGA ACA TCA AAG GAG GTG TAT GAC CAT GCC AGG AGG 1198
313 Phe Asp Ile Lys Arg Thr Ser Lys Glu Val Tyr Asp His Ala Arg Arg 328
1199 GCT CTG TAC AAT GCT GGC GTT GTG GAC CTC GTT TCA AGA AGC AAC CAG 1246
329 Ala Leu Tyr Asn Ala Gly Val Val Asp Leu Val Ser Arg Ser Asn Gln 344
1247 AGC CCT TCA CAC TCG CCT CTG AAG TCC TCA GAA AGC AGC ATG AAC TGC 1294
345 Ser Pro Ser His Ser Pro Leu Lys Ser Ser Glu Ser Ser Met Asn Cys 360
1295 AGC AGC TGC GAG GGC CTC AGC TGC CAG CAG ACC CGG GTG CTG CAG GAG 1342
361 Ser Ser Cys Glu Gly Leu Ser Cys Gln Gln Thr Arg Val Leu Gln Glu 376
1343 AAG CTA CGC AAG CTG AAG GAA GCC ATG CTG TGC ATG GTG TGC TGC GAG 1390
377 Lys Leu Arg Lys Leu Lys Glu Ala Met Leu Cys Met Val Cys Cys Glu 392
1391 GAG GAG ATC AAC TCC ACC TTC TGT CCC TGT GGC CAC ACT GTG TGC TGT 1438
393 Glu Glu Ile Asn Ser Thr Phe Cys Pro Cys Gly His Thr Val Cys Cys 408
1439 GAG AGC TGC GCC GCC CAG CTA CAG TCA TGT CCC GTC TGC AGG TCG CGT 1486
409 Glu Ser Cys Ala Ala Gln Leu Gln Ser Cys Pro Val Cys Arg Ser Arg 424
1487 GTG GAG CAT GTC CAG CAC GTC TAT CTG CCA ACG CAC ACC AGT CTT CTC 1534
425 Val Glu His Val Gln His Val Tyr Leu Pro Thr His Thr Ser Leu Leu 440
1535 AAT CTG ACT GTA ATC TAA TCT GTT GTG CTT TTG TTG GAC TTG GCA TGT 1582
441 Asn Leu Thr Val Ile *** 446
1583 TTC CAT GAA CTG CAC TAT TAT AAA CTA TTA AAA TGA TAG ATT GTG GAG 1630
1631 AAA GTA ATT ATT CCA ACA CCC ATC TGC CAT GCG ATG TTA AAA AAA AAA 1678
1679 AAA AAA A 1685
D.Blastp result
Query=PP5242 albumen (445 amino acid)
>SP_IN：Q24440 Q24440 drosophila melanogaster(fruit fly).(cdnal)
protein 4.1 homologue(coracle).11/1999
Length=1698 amino acid
Score value=125bits (312), predicated value=4e-28
Homogeny=82/284 (28%), similarity=138/284 (47%), breach=8/284 (2%)
Query：2 LCYVTRPDAVLMEVEVEAKANGEDCLNQVCRRLGIIEVDYFGLQFTGSKGESLWLNLRNR 61
L VT D L++V ++ KA G D +N +C L +IE DYFGL + WL+L
Sbjct：33 LARVTLLDGSLLDVSIDRKAIGRDVINSICAGLNLIEKDYFGLTYETPTDPRTWLDLEKP 92
Query：62 ISQQMDGLAPYRLKLRVKFF-VEPHLILQEQTRHIFFLHIKEALLAGHLLCSPEQAVELS 120
+S + + L VKF+ EP + ++ TR+ L ++ +L G L C+ L
Sbjct：93 VS-KFFRTDTWPLTFAVKFYPPEPSQLKEDITRYHLCLQVRNDILEGRLPCTFVTHALLG 151
Query：121 ALLAQTKFGDYNQN---TAKYNYEELCAKELSSATLNSIVAKHKELEGTSQASAEYQVLQ 177
+ L Q++ GDY+ T Y+ A ++ + ++ HK +G S A AE L+
Sbjct：152 SYLVQSEMGDYDAEEMPTRAYLKDFKIAPNQTAELEDKVMDLHKTHKGQSPAEAELHYLE 211
Query：178 IVSAMENYGIEWHSVRDSEGQKLLIGVGPEGISICKDDFSPINRIAYPVVQMATQSGKNV 237
+ YG++ H +DSEG +++GV G+ + +D INR A+P + + +
Sbjct：212 NAKKLAMYGVDLHPAKDSEGVDIMLGVCASGLLVYRDKLR-INRFAWPKILKISYKRHHF 270
Query：238 YLTVTKESGNS I--VLLFKMISTRAASGLYRAITETHAFYRCDT 279
Y+ + + FK+ + RAA L+++ E H F+R T
Sbjct：271 YIKIRPGEFEQYESTIGFKLANHRAAKKLWKSCVEHHTFFRLMT 314
9.PP5656 albumen
A: nucleotide sequence (SEQ ID NO:25) length: 1373bp
TTCCGCAATT TCTACCTGAC CTTCCTGGAG TACGATGGGA ACCTGCTGCG GAGAGAGCTC 60
TTTGTGTGCC CCAGCCAGCC CCCACCTGGT GCTGAGCAGT TGCAGCAGGC CCTGGCACAA 120
CTGGACGAGG AAGACCCCTG CTTTGAGTTC CGGCAGCAGC AGCTCACTGT GCACCGTGTG 180
CATGTCACTT TCCTGCCCCA TGAACCGCCA CCCCCCCGGC CTCACGATGT CACCCTTGTG 240
GCCCAGCTGT CCATGGACCG GCTGCAGATG TTGGAAGCCC TGTGCAGGCA CTGGCCTGGC 300
CCCATGAGCC TGGCCTTGTA CCTGACAGAC GCAGAAGCTC AGCAGTTCCT GATTTCGGTC 360
GAGGCCTCAC CAGTGCTTGC TGCCCGGCAG GACGTGGCCT ACCATGTGGT GTACCGTGAG 420
GGCCCCTATA CCCCGTCAAC CAGCTTCGCA ACGTGGCCTT GGCCAGGCCC TCACGCCTTA 480
CGTCTTCCTC AGTGACATTG ACTTCCTGCC TGCCTATTCT CTCTACGACT ACCTCAGGGC 540
CTCCATTGAG CAGCTGGGGC TGGGCAGCCG GCGCAAGGCA GCACTGGTGG TGCCGGCATT 600
CGAGACCCTG CGCTACCGCT TCAGCTTCCC CCATTCCAAG GTGGAGCTGT TGGCCTTGCT 660
GGATGCGGGC ACTCTCTACA CCTTCAGGTA CCACGAGTGG CCCCGAGGCC ACGCACCCAC 720
AGACTATGCC CGCTGGCGGG AGGCTCAGGC CCCGTACCGT GTGCAATGGG CGGCCAACTA 780
TGAACCCTAC GTGGTGGTGC CACGAGACTG TCCCCGCTAT GATCCTCGCT TTGTGGGCTT 840
CCGCTGGAAC AAAGTGGCCC ACATTGTGGA GCTGGATGCC CAGGAATATG AGCTCCTGGT 900
GCTGCCCGAG GCCTTCACCA TCCATCTGCC CCACGCTCCA AGCCTGGACA TCTCCGCTTC 960
CGCTCCAGCC CCACCTATCG TGACTGCCTC CAGGCCCTCA AGGACGAATT CCACCAGGAC 1020
TTGTCCCGCC ACCATGGGGC TGCTGCCCTC AAATACCTCC CAGCCCTGCA GCAGCCCCAG 1080
AGCCCTGCCC GAGGCTGAGG CTGGGCCGGC GCTGCCCCTC ATCTTAGCAT TGGGCAGACA 1140
CCAGGGCAAC CTGCCCTCCG CCATCCCTGC TATTTAAATT ATTTAAGGTC TCTGGGAAGG 1200
GCTGGGGCAG AGCATCTGTG GGGTGGGGTC TTCCCCTTGC TGCTATTGTA TGGCTGGGGA 1260
CTGGTCTCTC TCTGCCCCAG CCAGTTTGGG GCTGGTTCCC CCATCTTGAA TTGTTTATCC 1320
CTTTTTCATA ATTAAAGTTT TAAAACATAA AAAAAAAAAA AAAAAAAAAA AAA 1373
B: aminoacid sequence (SEQ ID NO:26) length: 257 amino acid
1 MWCTVRAPIP RQPASQRGLG QALTPYVFLS DIDFLPAYSL YDYLRASIEQ LGLGSRRKAA
61 LVVPAFETLR YRFSFPHSKV ELLALLDAGT LYTFRYHEWP RGHAPTDYAR WREAQAPYRV
121 QWAANYEPYV VVPRDCPRYD PRFVGFGWNK VAHIVELDAQ EYELLVLPEA FTIHLPHAPS
181 LDISASAPAP PIVTASRPSR TNSTRTCPAT MGLLPSNTSQ PCSSPRALPE AEAGPALPLI
241 LALGRHQGNL PSAIPAI
C. Nucleotide and amino acid composite sequence (SEQ ID NO:27)
Clone number: PP5656
Start code: 404 ATG stop coding: 1177 TAA
Protein molecular weight: 28557
1 T TCC GCA ATT TCT ACC TGA CCT TCC TGG AGT ACG ATG GGA ACC TGC 46
47 TGC GGA GAG AGC TCT TTG TGT GCC CCA GCC AGC CCC CAC CTG GTG CTG 94
95 AGC AGT TGC AGC AGG CCC TGG CAC AAC TGG ACG AGG AAG ACC CCT GCT 142
143 TTG AGT TCC GGC AGC AGC AGC TCA CTG TGC ACC GTG TGC ATG TCA CTT 190
191 TCC TGC CCC ATG AAC CGC CAC CCC CCC GGC CTC ACG ATG TCA CCC TTG 238
239 TGG CCC AGC TGT CCA TGG ACC GGC TGC AGA TGT TGG AAG CCC TGT GCA 286
287 GGC ACT GGC CTG GCC CCA TGA GCC TGG CCT TGT ACC TGA CAG ACG CAG 334
335 AAG CTC AGC AGT TCC TGA TTT CGG TCG AGG CCT CAC CAG TGC TTG CTG 382
383 CCC GGC AGG ACG TGG CCT ACC ATG TGG TGT ACC GTG AGG GCC CCT ATA 430
1 Met Trp Cys Thr Val Arg Ala Pro Ile 9
431 CCC CGT CAA CCA GCT TCG CAA CGT GGC CTT GGC CAG GCC GTC ACG CCT 478
10 Pro Arg Gln Pro Ala Ser Gln Arg Gly Leu Gly Gln Ala Leu Thr Pro 25
479 TAC GTC TTC CTC AGT GAC ATT GAC TTC CTG CCT GCC TAT TCT CTC TAC 526
26 Tyr Val Phe Leu Ser Asp Ile Asp Phe Leu Pro Ala Tyr Ser Leu Tyr 41
527 GAC TAC CTC AGG GCC TCC ATT GAG CAG CTG GGG CTG GGC AGC CGG CGC 574
42 Asp Tyr Leu Arg Ala Ser Ile Glu Gln Leu Gly Leu Gly Ser Arg Arg 57
575 AAG GCA GCA CTG GTG GTG CCG GCA TTC GAG ACC CTG CGC TAC CGC TTC 622
58 Lys Ala Ala Leu Val Val Pro Ala Phe Glu Thr Leu Arg Tyr Arg Phe 73
623 AGC TTC CCC CAT TCC AAG GTG GAG CTG TTG GCC TTG CTG GAT GCG GGC 670
74 Ser Phe Pro His Ser Lys Val Glu Leu Leu Ala Leu Leu Asp Ala Gly 89
671 ACT CTC TAC ACC TTC AGG TAC CAC GAG TGG CCC CGA GGC CAC GCA CCC 718
90 Thr Leu Tyr Thr Phe Arg Tyr His Glu Trp Pro Arg Gly His Ala Pro 105
719 ACA GAC TAT GCC CGC TGG CGG GAG GCT CAG GCC CCG TAC CGT GTG CAA 766
106 Thr Asp Tyr Ala Arg Trp Arg Glu Ala Gln Ala Pro Tyr Arg Val Gln 121
767 TGG GCG GCC AAC TAT GAA CCC TAC GTG GTG GTG CCA CGA GAC TGT CCC 814
122 Trp Ala Ala Asn Tyr Glu Pro Tyr Val Val Val Pro Arg Asp Cys Pro 137
815 CGC TAT GAT CCT CGC TTT GTG GGC TTC GGC TGG AAC AAA GTG GCC CAC 862
138 Arg Tyr Asp Pro Arg Phe Val Gly Phe Gly Trp Asn Lys Val Ala His 153
863 ATT GTG GAG CTG GAT GCC CAG GAA TAT GAG CTC CTG GTG CTG CCC GAG 910
154 Ile Val Glu Leu Asp Ala Gln Glu Tyr Glu Leu Leu Val Leu Pro Glu 169
911 GCC TTC ACC ATC CAT CTG CCC CAC GCT CCA AGC CTG GAC ATC TCC GCT 958
170 Ala Phe Thr Ile His Leu Pro His Ala Pro Ser Leu Asp Ile Ser AIa 185
959 TCC GCT CCA GCC CCA CCT ATC GTG ACT GCC TCC AGG CCC TCA AGG ACG 1006
186 Ser Ala Pro Ala Pro Pro Ile Val Thr Ala Ser Arg Pro Ser Arg Thr 201
1007 AAT TCC ACC AGG ACT TGT CCC GCC ACC ATG GGG CTG CTG CCC TCA AAT 1054
202 Asn Ser Thr Arg Thr Cys Pro Ala Thr Met Gly Leu Leu Pro Ser Asn 217
1055 ACC TCC CAG CCC TGC AGC AGC CCC AGA GCC CTG CCC GAG GCT GAG GCT 1102
218 Thr Ser Gln Pro Cys Ser Ser Pro Arg Ala Leu Pro Glu Ala Glu Ala 233
1103 GGG CCG GCG CTG CCC CTC ATC TTA GCA TTG GGC AGA CAC CAG GGC AAC 1150
234 Gly Pro Ala Leu Pro Leu Ile Leu Ala Leu Gly Arg His Gln Gly Asn 249
1151 CTG CCC TCC GCC ATC CCT GCT ATT TAA ATT ATT TAA GGT CTC TGG GAA 1198
250 Leu Pro Ser Ala Ile Pro Ala Ile *** 258
1199 GGG CTG GGG CAG AGC ATC TGT GGG GTG GGG TCT TCC CCT TGC TGC TAT 1246
1247 TGT ATG GCT GGG GAC TGG TCT CTC TCT GCC CCA GCC AGT TTG GGG CTG 1294
1295 GTT CCC CCA TCT TGA ATT GTT TAT CCC TTT TTC ATA ATT AAA GTT TTA 1342
1343 AAA CAT AAA AAA AAA AAA AAA AAA AAA AAA A 1373
D.Blastp result
Query=PP5656 albumen (257 amino acid)
>SP_IN：Q21389 Q21389 caenorhabditis elegans.k09c8.4 protein.11/1999
Length=622 amino acid
Score value=104bits (256), predicated value=8e-22
Homogeny=65/166 (39%), similarity=93/166 (55%), breach=17/166 (10%)
Query：26 YVFLSDIDFLPAYSLYDYLRASIEQLGLGSRRKAALVVPAFET----LRYRFSFPHSKVE 81
Y+ ++D+DF+ L DY I+Q G ++K LV+PA E LR S S+ +
Sbjct：427 YILMTDVDFVV---LGDY-GTIIDQTG-NLKQKEVLVIPALEMTYPQLRLNLSNFLSRKD 481
Query：82 LLA--LLDAGTLYTFRYHEWPRGHAPTDYARWREAQAPY----RVQWAANYEPYVVVPRD 135
L+ LL+ T+ TFR WP H PT+ ++W ++ Y V + NYEPY V+ ++
Sbjct：482 LVIEHLLNK-TIQTFRETIWPSSHVPTNISKWIKSNRTYMVAQNVNYEKNYEPYFVIKKE 540
Query：136 -CPRYDPRFVGFGWNKVAHIVELDAQEYELLVLPEAFTIHLPHAPS 180
CP YD RF GFGWNKV H+++L Y+ LV P +F IH H S
Sbjct：541 ECPFYDQRFGGFGWNKVTHVMQLKMMNYKFLVSPTSFMIHQNHNAS 586
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. isolating human polypeptides with cancer suppressing function, it is characterized in that, it contains the aminoacid sequence that is selected from down group: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, and described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.
2. polypeptide as claimed in claim 1, it is characterized in that this polypeptid acid sequence is selected from down group: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQID NO:26.
3. isolating polynucleotide is characterized in that, it is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3, it is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:26.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:
Coding region sequence or the full length sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it is a kind of host cell that is selected from down group:
(a) host cell that transforms or transduce with the described carrier of claim 6;
(b) host cell that transforms or transduce with the described polynucleotide of claim 3.
8. the preparation method of the polypeptide of the people's protein-active with cancer suppressing function is characterized in that this method comprises:
(a) have under the proteic condition of people of cancer suppressing function suitable the expression, cultivate the described host cell of claim 7;
(b) isolate polypeptide from culture, described polypeptide forms inhibited to the clone of the hepatoma cell line 7721 of cultivation.
9. energy and the described human polypeptides specificity bonded antibody with cancer suppressing function of claim 1, wherein said human polypeptides forms inhibited to the clone of the hepatoma cell line 7721 of cultivating.
10. a pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
CNB001157450A 2000-05-18 2000-05-18 New human protein with the function of inhibiting cancer cell growth and its encoding sequence Expired - Fee Related CN1193041C (en)

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