CN1169958C - Human protein able to suppress growth of cancer cells and its coding sequence - Google Patents

Abstract

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

Description

The proteic polynucleotide of people that coding has anticancer growth function

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.

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.

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

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

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated protein polypeptide with cancer suppressing function is provided, and it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:14; 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.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned protein polypeptide with cancer suppressing function of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:14.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 IDNO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15.

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.

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.Be example with PP565 albumen (in this application, its clone numbering is adopted in proteinic name), 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 IDNO:3.Be example with PP712 albumen (in this application, its clone numbering is adopted in proteinic name), the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:6 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:5, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:6.Have the albumen of cancer suppressing function for other, can the rest may be inferred.Have the albumen of cancer suppressing function for other, can the rest may be inferred.

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

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

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

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ IDNO:2.

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

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

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

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

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

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) 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, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

With the also available labelled with radioisotope of the protein bound monoclonal antibody of the people with cancer suppressing function, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody among the present invention can be used for treating or prevents and the relevant disease of people's albumen with cancer suppressing function.The antibody that gives suitable dosage can stimulate or block proteic generation of the people with cancer suppressing function or activity.

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

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

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

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

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

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

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

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

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

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

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

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

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

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

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

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

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

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

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

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

PP565, PP712, PP1143, PP3241, PP3501 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-mentioned 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)
			PP565	2 12 7	20 17 16
PP712	7 3 8	21 18 19
			PP1143	9 10 6	42 40 56
PP3241	2 0 0	12 18 20
			PP3501	0 0 0	28 35 25

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, do not obtain full length cDNA sequence yet, the design primer checks order once more, up to obtaining full length sequence (SEQ ID NO:1,4,7,10,13).

Embodiment 2: PGR obtains full-length gene 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.Carry out reverse transcription reaction with MMLV-RT-Superscript II (GIBCO BRL) ThermoScript II at 42 ℃, obtain placenta cDNA.Utilize the different primer of commentaries on classics (as shown in the table) of each gene, by 90 ℃ of 1 circulations in 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute, totally 35 circulations; 72 ℃ 10 minutes, pcr amplification is carried out in 1 circulation, obtains to contain the amplified production of each protein gene of complete open reading frame sequence.Amplified production is through sequence verification, and the sequence that records with embodiment 1 conforms to, and changes amplified production over to host cell with routine techniques subsequently, to obtain recombinant protein.

Gene specific primer

Clone's title	Special primer 1 (5 ' → 3 ')	Special primer 2 (5 ' → 3 ')
			PP565	TGGGCTTTGGTGGAAACCGAATG	CTGTGGCTTCACCCAGTCCCTCT
PP712	TTTCCCATGCTCCCTGCCTTTCC	AGCAGGGAAGGGGGATACCTGGG
			PP1143	GGCTCTGAACCAAAGGAGCAGGA	GGGCTCTTGGACCACTGCCTCCT
PP3241	TTCAAGCCAGTGAACAACGCGGA	GGGGCTGTAGGAGATGGGTTGGC
			PP3501	CGGGGTGACTGGTATACCCCCAA	AACCCTCAGGCAAGAGGCTGCAC

Embodiment 3:cDNA cloned sequence is analyzed

1.PP565

A: nucleotide sequence: (SEQ ID NO:1) length: 1932bp

1 CTAATATTGA CTTAGACTCA TTTCAAAGTC CTAACATGAT CTTCAGCCTT

51 CCTTTCAGAG AAACTAATTC TCTAAATATG TATTCCTAGA CCCACTATCA

101 TTGTCTTTGT ACTGAATGAT TATTGACAGT TTTTTGGATT TCTAGAGACC

151 TCTCCTGACA GAGAAATATC CTGGTCCCTA GGAAGCATTC TCTGTGATTT

201 TGGTTGCTAT TGGTCTGTTA CTGGGCTTTG GTGGAAACCG AATGTTTGAC

251 TATGGATCAT CAAGTCACCA TGCAACCTGA ACTACCTATC ATGAACTGGG

301 TGCTTTCTGA CCTATCTAGC CATAAAGTGG GTCATGCACA GCAGCATTCC

351 ATCATCAAAT GAAAGTATAC TTGATCGGGC TTGAGAAGGT CCTGAAGGCA

401 CAAATAAGTT ACATGAAGAA GTGGCTCAAA TGCCCATGGT CTCCACTCCT

451 GCCACCCTGC CTCCTCTCCC CTAGCCTGCA CCAGTGGCTT CATGGGGAGT

501 TCCCTATGAT TAGTTGACAG AGGAAGAGAA GACTAAGGCC TGGTTCACAG

551 ATGATTCTGC ATGATATGCA GGCACTACCT GAAAGTAGAC CACTGCATTA

601 CAACCCTTTT CTAGGACATC CCTGAAGGAC AGCAGTGAAG GGAAATCTTC

651 CCAGTGGGCA GAATTTCGAG CAGTGCACCT GGTTGTGTAC TTTGCCTGGA

701 AGGAGAAATG TCCAGATGTG CGATTATATA CTGATTCATT GGCTGTAGCC

751 AGTGGTTTGG CTGTATGGTC AGGGACTTGG AAGAAGTATG ATTGGAAAAT

801 TGGTGACAAA GAAATTTGGG GAAGAGGTAA GTGGATGGAC CTTTCTGAGT

851 GATTGAAAAC TGAAGATATT TGTATCTCAT GTGAGTGCTC ACCAATGGGT

901 GACCTTGGCG GAGGAGGATT TCAATAATCA AGTGGATAGG ATGACCTGTT

951 CTGTGGACAT CACTCAGCGT CTTTCCCCAG CCACCTCTCT TATTCCCCAG

1001 TGGACCCATG AACTATGTGG TCATGGTGGC AAGGATGGAG GTTACACATG

1051 GGCTCAGCAA CATGGACTTC CACTCACCAA GGCTGACCTG GCTACCGCCA

1101 CTGCTGAGTG CCCAATTTGC CAGCAGCGGA TACCAACACT GAACCCTCGA

1151 TATGGCGCCA TTCCTCAGAG TGATCAGCCA GCTACCTGGT GGCAGGTTGA

1201 TTATATTGGA TCTCTTCCAT CATAGCAAGG GCAGAGGTTT GTCCTCACTG

1251 GAACAGACAC TTACTCTGGA TATGGGTTTG CCTATCCTGC ATGCAATGTT

1301 TCTGCCAAGA CTCCATCCAT GGATTCATGG AATGCATTAT CTGTTGTGGG

1351 AAGTCAGGGA CCCTGAATAG AGGGACTGGG TGAAGCCACA GCAGAAGAAC

1401 ATAAATTGTG AAGATTTCAT GGACATTTAT TAATTCCCTA AATTAATACT

1451 TTTATAATTT CTTACGCCTG TCTTTACTGC AGTCTCTGAA CATAAATTGT

1501 GAAGATTTCA TGGACATTTC TGACTTCTCA ATCAATACTC TTATAATTTC

1551 CTATGCCTGT CTTTACTTTA ATCTCTTAAT CCCATCATCT TTGTAAAGTA

1601 AGGATGTATG TCACCTCAGG ACCCTGTGAT GATTATGTTA TCTGTATAAA

1651 TTGTTTGTAA AACATGCGTG TTTGAACAAT ATGAAATCTG GGCATCCTAA

1701 AAGAACAGGA TAACAGTGAT TTTCAGGGAA CAAGGGAAAT AACCATAAAG

1751 TCTGACTGCC CATTTGCCTT GTGATATTTT GTTGCCCTTG AAGCATGTGA

1801 TTTCTGTGAC CCACACCCTA TTCGTACACC CCTCCCCTTT TGAAATCCAT

1851 AATAAAAACT TGCTGGTTTT GTGGCTCAGG GGGCATCCCC AAAAAAAAAA

1901 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA

B: aminoacid sequence: (SEQ ID NO:2) length: 94 amino acid

1 MTCSVDITQP LSPATSLIPQ WTHELCGHGG KDGGYTWAQQ HGLPLTKADL

51 ATATAECPIC QQRIPTLNPR YGAIPQSDQP ATWWQVDYIG SLPS

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

Clone number and protein name: PP565

Start code: 941 ATG stop coding: 1225 TAG

Protein molecular weight: 10188.93

1 C TAA TAT TGA CTT AGA CTC ATT TCA AAG TCC TAA CAT GAT CTT CAG 46

47 CCT TCC TTT CAG AGA AAC TAA TTC TCT AAA TAT GTA TTC CTA GAC CCA 94

95 CTA TCA TTG TCT TTG TAC TGA ATG ATT ATT GAC AGT TTT TTG GAT TTC 142

143 TAG AGA CCT CTC CTG ACA GAG AAA TAT CCT GGT CCC TAG GAA GCA TTC 190

191 TCT GTG ATT TTG GTT GCT ATT GGT CTG TTA CTG GGC TTT GGT GGA AAC 238

239 CGA ATG TTT GAC TAT GGA TCA TCA AGT CAC CAT GCA ACC TGA ACT ACC 286

287 TAT CAT GAA CTG GGT GCT TTC TGA CCT ATC TAG CCA TAA AGT GGG TCA 334

335 TGC ACA GCA GCA TTC CAT CAT CAA ATG AAA GTA TAC TTG ATC GGG CTT 382

383 GAG AAG GTC CTG AAG GCA CAA ATA AGT TAC ATG AAG AAG TGG CTC AAA 430

431 TGC CCA TGG TCT CCA CTC CTG CCA CCC TGC CTC CTC TCC CCT AGC CTG 478

479 CAC CAG TGG CTT CAT GGG GAG TTC CCT ATG ATT AGT TGA CAG AGG AAG 526

527 AGA AGA CTA AGG CCT GGT TCA CAG ATG ATT CTG CAT GAT ATG CAG GCA 574

575 CTA CCT GAA AGT AGA CCA CTG CAT TAC AAC CCT TTT CTA GGA CAT CCC 622

623 TGA AGG ACA GCA GTG AAG GGA AAT CTT CCC AGT GGG CAG AAT TTC GAG 670

671 CAG TGC ACC TGG TTG TGT ACT TTG CCT GGA AGG AGA AAT GTC CAG ATG 718

719 TGC GAT TAT ATA CTG ATT CAT TGG CTG TAG CCA GTG GTT TGG CTG TAT 766

767 GGT CAG GGA CTT GGA AGA AGT ATG ATT GGA AAA TTG GTG ACA AAG AAA 814

815 TTT GGG GAA GAG GTA AGT GGA TGG ACC TTT CTG AGT GAT TGA AAA CTG 862

863 AAG ATA TTT GTA TCT CAT GTG AGT GCT CAC CAA TGG GTG ACC TTG GCG 910

911 GAG GAG GAT TTC AAT AAT CAA GTG GAT AGG ATG ACC TGT TCT GTG GAC 958

1 Met Thr Cys Ser Val Asp 6

959 ATC ACT CAG CCT CTT TCC CCA GCC ACC TCT CTT ATT CCC CAG TGG ACC 1006

7 Ile Thr Gln Pro Leu Ser Pro Ala Thr Ser Leu Ile Pro Gln Trp Thr 22

1007 CAT GAA CTA TGT GGT CAT GGT GGC AAG GAT GGA GGT TAC ACA TGG GCT 1054

23 His Glu Leu Cys Gly His Gly Gly Lys Asp Gly Gly Tyr Thr Trp Ala 38

1055 CAG CAA CAT GGA CTT CCA CTC ACC AAG GCT GAC CTG GCT ACC GCC ACT 1102

39 Gln Gln His Gly Leu Pro Leu Thr Lys Ala Asp Leu Ala Thr Ala Thr 54

1103 GCT GAG TGC CCA ATT TGC CAG CAG CGG ATA CCA ACA CTG AAC CCT CGA 1150

55 Ala Glu Cys Pro Ile Cys Gln Gln Arg Ile Pro Thr Leu Asn Pro Arg 70

1151 TAT GGC GCC ATT CCT CAG AGT GAT CAG CCA GCT ACC TGG TGG CAG GTT 1198

71 Tyr Gly Ala Ile Pro Gln Ser Asp Gln Pro Ala Thr Trp Trp Gln Val 86

1199 GAT TAT ATT GGA TCT CTT CCA TCA TAG CAA GGG CAG AGG TTT GTC CTC 1246

87 Asp Tyr Ile Gly Ser Leu Pro Ser *** 95

1247 ACT GGA ACA GAC ACT TAC TCT GGA TAT GGG TTT GCC TAT CCT GCA TGC 1294

1295 AAT GTT TCT GCC AAG ACT CCA TCC ATG GAT TCA TGG AAT GCA TTA TCT 1342

1343 GTT GTG GGA AGT CAG GGA CCC TGA ATA GAG GGA CTG GGT GAA GCC ACA 1390

1391 GCA GAA GAA CAT AAA TTG TGA AGA TTT CAT GGA CAT AAA TTA ATT CCC 1438

1439 TAA ATT AAT ACT TTT ATA ATT TCT TAC GCC TGT CTT TAC TGC AGT CTC 1486

1487 TGA ACA TAA ATT GTG AAG ATT TCA TGG ACA TTT CTG ACT TCT CAA TCA 1534

1535 ATA CTC TTA TAA TTT CCT ATG CCT GTC TTT ACT TTA ATC TCT TAA TCC 1582

1583 CAT CAT CTT TGT AAA GTA AGG ATG TAT GTC ACC TCA GGA CCC TGT GAT 1630

1631 GAT TAT GTT ATC TGT ATA AAT TGT TTG TAA AAC ATG CGT GTT TGA ACA 1678

1679 ATA TGA AAT CTG GGC ATC CTA AAA GAA CAG GAT AAC AGT GAT TTT CAG 1726

1727 GGA ACA AGG GAA ATA ACC ATA AAG TCT GAC TGC CCA TTT GCC TTG TGA 1774

1775 TAT TTT GTT GCC CTT GAA GCA TGT GAT TTC TGT GAC CCA CAC CCT ATT 1822

1823 CGT ACA CCC CTC CCC TTT TGA AAT CCA TAA TAA AAA CTT GCT GGT TTT 1870

1871 GTG GCT CAG GGG GCA TCC CCA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1918

1919 AAA AAA AAA AAA AA 1932

D:Blastp result

Query=PP565AA (94 amino acid)

>SP_VI：O02711 O02711 murine endogenous retrovirus.pro-pol-dutpase

polyprotein(fragment).5/1999

Length=1182

Score value=155bits (387), predicated value=1e-37

Homogeny=70/94 (74%), similarity=78/94 (82%), breach=1/94 (1%)

Query：1 MTCSVDITQPLSPATSLIPQWTHELCGHGGKDGGYTWAQQHGLPLTKADLATATAECPIC 60

MT SVD +Q LSPA +I QW HE GHGG+DGGY WAQQHGLPLTKADLATA A+C IC

Sbjct：753 MTRSVD-SQTLSPAIPVIAQWAHEQSGHGGRDGGYPWAQQHGLPLTKADLATAAADCQIC 811

Query：61 QQRIPTLNPRYGAIPQSDQPATWWQVDYIGSLPS 94

QQ+ PTL+PRYG I P+DQPATWWQVDY+G LPS

Sbjct：812 QQQKPTLSPRYGTIPRGDQPATWWQVDYVGPLPS 845

2.PP712

A: nucleotide sequence: (SEQ ID NO:4) length: 1553bp

1 CCTGGCTCTG CCCACAATCA CCAGGGCAGC TTGGTAAGGA TGAAGATGAC

51 AAGGTCCATC CTCAGACTGA TGGAATCATG AACGAGCATC TTAGGATAGT

101 GGCAGGGCTG GATGATGGCA TGTTTCCCAT GCTCCCTGCC TTTCCCAAGT

151 CCTGCTGAGT ACAGACCCAA CTGCCCCTAG GTTGGTCCTG CCTCTAGCCT

201 TCCCTAGAGT CTGGGGCGAT GGAAGCCTTG TGTGTGTGCT CACTCATGCC

251 TCTTCATTGG TCCTGGATCC TTACAGCTTC CCATCTGGCT GCTTTTTTCC

301 AGGGCTTCCG AAGTCTGGAA GACATCCGCA GCCAGGCCTC CCTGACAACC

351 CAGCAGGCCA TCGGCCTGAA GCATTACAGT GACTTCCTGG AACGTATGCC

401 CAGGGAGGAG GCTACAGAGA TTGAGCAGAC AGTCCAGAAA GCAGCCCAGG

451 CCTTTAACTC TGGGCTGCTG TGTGTGGCAT GTGGTTCATA CCGACGGGGA

501 AAGGCGACCT GTGGTGATGT CGACGTGCTC ATCACTCACC CAGATGGCCG

551 GTCCCACCGG GGTATCTTCA GCCGCCTCCT TGACAGTCTT CGGCAGGAAG

601 GGTTCCTCAC AGATGACTTG GTGAGCCAAG AGGAGAATGG TCAGCAACAG

651 AAGTACTTGG GGGTGTGCCG GCTCCCAGGG CCAGGGCGGC GGCACCGGCG

701 CCTGGACATC ATCGTGGTGC CCTATAGCGA GTTTGCCTGT GCCCTGCTCT

751 ACTTCACCGG CTCTGCACAC TTCAACCGCT CCATGCGAGC CCTGGCCAAA

801 ACCAAGGGCA TGAGTCTGTC AGAACATGCC CTCAGCACTG CTGTGGTCCG

851 GAACACCCAT GGCTGCAAGG TGGGGCCTGG CCGAGTGCTG CCCACTCCCA

901 CTGAGAAGGA TGTCTTCAGG CTCTTAGGCC TCCCCTACCG AGAACCTGCT

951 GAGCGGGACT GGTGACCCAT GGCTGGGGGT GCTGAGGAGA GCCGAGTTGG

1001 ACTGGCTACC CCTCCTGGCC ACCCAGTACT CCCTCCAGCC TCAGCTGGCT

1051 GAACCTCGCC GCTCCAACCA CCAGCTTCCT CAGCGAGCAG GGCCCAGGGC

1101 TCTGGGCCTG AAGCAAGAGC CAGCCCGGCT CCCAGTGTCT GCCCGGCTCC

1151 CAGTGTCTGC CCAGCCCTCT CCCAGACAGG AGCAGGCTGC CACCCCTTCT

1201 ACCTCACCAC TGCCCCTCGA AGAATTTTGC AAATGGCCCC TTGCCCCATT

1251 TTAAGCAGGA GCAGGTGGCT GGTTTGAAGC CCCAGGTATC CCCCTTCCCT

1301 GCTATGGGAA AGGCCAAGCT GCTGGGTGGG GACAGAAGCT GCAGGGGAGA

1351 GGGAAGCAGC CGTGCTGTCA ACATCATCCG GCACCCTCTG GGGTAGGAGA

1401 ACAGCCATTC CACATGTGTT TCCCTCTATC CGTCCTGCTT CCTGGGCAGC

1451 TGGTGGTGCT GGGAATGGGG TGCCCCAGCC TTGGTGAGGA CAGTGTTGGG

1501 AGGCCCAGGG GCCCAGTAAA GTGCATTTGA CATTGAAAAA AAAAAAAAAA

1551 AAA

B: aminoacid sequence: PP712 (SEQ ID NO:5) length: 248 amino acid

1 MEALCVCSLM PLHWSWILTA SHLAAFFQGF RSLEDIRSQA SLTTQQAIGL

51 KHYSDFLERM PREEATEIEQ TVQKAAQAFN SGLLCVACGS YRRGKATCGD

101 VDVLITHPDG RSHRGIFSRL LDSLRQEGFL TDDLVSQEEN GQQQKYLGVC

151 RLPGPGRRHR RLDIIVVPYS EFACALLYFT GSAHFNRSMR ALAKTKGMSL

201 SEHALSTAVV RNTHGCKVGP GRVLPTPTEK DVFRLLGLPY REPAERDW

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

Clone number and protein name: PP712

Start code: 219 ATG stop coding: 965 TGA

Protein molecular weight: 27765.31

1 CC TGG CTC TGC CCA CAA TCA CCA GGG CAG CTT GGT AAG GAT GAA GAT 47

48 GAC AAG GTC CAT CCT CAG ACT GAT GGA ATC ATG AAC GAG CAT CTT AGG 95

96 ATA GTG GCA GGG CTG GAT GAT GGC ATG TTT CCC ATG CTC CCT GCC TTT 143

144 CCC AAG TCC TGC TGA GTA CAG ACC CAA CTG CCC CTA GGT TGG TCC TGC 191

192 CTC TAG CCT TCC CTA GAG TCT GGG GCG ATG GAA GCC TTG TGT GTG TGC 239

1 Met Glu Ala Leu Cys Val Cys 7

240 TCA CTC ATG CCT CTT CAT TGG TCC TGG ATC CTT ACA GCT TCC CAT CTG 287

8 Ser Leu Met Pro Leu His Trp Ser Trp Ile Leu Thr Ala Ser His Leu 23

288 GCT GCT TTT TTC CAG GGC TTC CGA AGT CTG GAA GAC ATC CGC AGC CAG 335

24 Ala Ala Phe Phe Gln Gly Phe Arg Ser Leu Glu Asp Ile Arg Ser Gln 39

336 GCC TCC CTG ACA ACC CAG CAG GCC ATC GGC CTG AAG CAT TAC AGT GAC 383

40 Ala Ser Leu Thr Thr Gln Gln Ala Ile Gly Leu Lys His Tyr Ser Asp 55

384 TTC CTG GAA CGT ATG CCC AGG GAG GAG GCT ACA GAG ATT GAG CAG ACA 431

56 Phe Leu Glu Arg Met Pro Arg Glu Glu Ala Thr Glu Ile Glu Gln Thr 71

432 GTC CAG AAA GCA GCC CAG GCC TTT AAC TCT GGG CTG CTG TGT GTG GCA 479

72 Val Gln Lys Ala Ala G1n Ala Phe Asn Ser Gly Leu Leu Cys Val Ala 87

480 TGT GGT TCA TAC CGA CGG GGA AAG GCG ACC TGT GGT GAT GTC GAC GTG 527

88 Cys Gly Ser Tyr Arg Arg Gly Lys Ala Thr Cys Gly Asp Val Asp Val 103

528 CTC ATC ACT CAC CCA GAT GGC CGG TCC CAC CGG GGT ATC TTC AGC CGC 575

104 Leu Ile Thr His Pro Asp Gly Arg Ser His Arg Gly Ile Phe Ser Arg 119

576 CTC CTT GAC AGT CTT CGG CAG GAA GGG TTC CTC ACA GAT GAC TTG GTG 623

120 Leu Leu Asp Ser Leu Arg Gln Glu Gly Phe Leu Thr Asp Asp Leu Val 135

624 AGC CAA GAG GAG AAT GGT CAG CAA CAG AAG TAC TTG GGG GTG TGC CGG 671

136 Ser Gln Glu Glu Asn Gly Gln Gln Gln Lys Tyr Leu Gly Val Cys Arg 151

672 CTC CCA GGG CCA GGG CGG CGG CAC CGG CGC CTG GAC ATC ATC GTG GTG 719

152 Leu Pro Gly Pro Gly Arg Arg His Arg Arg Leu Asp Ile Ile Val Val 167

720 CCC TAT AGC GAG TTT GCC TGT GCC CTG CTC TAC TTC ACC GGC TCT GCA 767

168 Pro Tyr Ser Glu Phe Ala Cys Ala Leu Leu Tyr Phe Thr Gly Ser Ala 183

768 CAC TTC AAC CGC TCC ATG CGA GCC CTG GCC AAA ACC AAG GGC ATG AGT 815

184 His Phe Asn Arg Ser Met Arg Ala Leu Ala Lys Thr Lys Gly Met Ser 199

816 CTG TCA GAA CAT GCC CTC AGC ACT GCT GTG GTC CGG AAC ACC CAT GGC 863

200 Leu Ser Glu His Ala Leu Ser Thr Ala Val Val Arg Asn Thr His Gly 215

864 TGC AAG GTG GGG CCT GGC CGA GTG CTG CCC ACT CCC ACT GAG AAG GAT 911

216 Cys Lys Val Gly Pro Gly Arg Val Leu Pro Thr Pro Thr Glu Lys Asp 231

912 GTC TTC AGG CTC TTA GGC CTC CCC TAC CGA GAA CCT GCT GAG CGG GAC 959

232 Val Phe Arg Leu Leu Gly Leu Pro Tyr Arg Glu Pro Ala Glu Arg Asp 247

960 TGG TGA CCC ATG GCT GGG GGT GCT GAG GAG AGC CGA GTT GGA CTG GCT 1007

248 Trp *** 249

1008 ACC CCT CCT GGC CAC CCA GTA CTC CCT CCA GCC TCA GCT GGC TGA ACC 1055

1056 TCG CCG CTC CAA CCA CCA GCT TCC TCA GCG AGC AGG GCC CAG GGC TCT 1103

1104 GGG CCT GAA GCA AGA GCC AGC CCG GCT CCC AGT GTC TGC CCG GCT CCC 1151

1152 AGT GTC TGC CCA GCC CTC TCC CAG ACA GGA GCA GGC TGC CAC CCC TTC 1199

1200 TAC CTC ACC ACT GCC CCT CGA AGA ATT TTG CAA ATG GCC CCT TGC CCC 1247

1248 ATT TTA AGC AGG AGC AGG TGG CTG GTT TGA AGC CCC AGG TAT CCC CCT 1295

1296 TCC CTG CTA TGG GAA AGG CCA AGC TGC TGG GTG GGG ACA GAA GCT GCA 1343

1344 GGG GAG AGG GAA GCA GCC GTG CTG TCA ACA TCA TCC GGC ACC CTC TGG 1391

1392 GGT AGG AGA ACA GCC ATT CCA CAT GTG TTT CCC TCT ATC CGT CCT GCT 1439

1440 TCC TGG GCA GCT GGT GGT GCT GGG AAT GGG GTG CCC CAG CCT TGG TGA 1487

1488 GGA CAG TGT TGG GAG GCC CAG GGG CCC AGT AAA GTG CAT TTG ACA TTG 1535

1536 AAA AAA AAA AAA AAA AAA 1553

D:Blastp result

Query=PP712[gene=PP712] (248 amino acid)

>SW：YA26_SCHPO Q09693 schizosaccharomyces pombe(fission yeast).

hypothetical dna polymerase beta-like protein c2f7.06c.

11/1995

Length=506

Score value=80.8bits (196), predicated value=1e-14

Homogeny=70/238 (29%), similarity=110/238 (45%), breach=17/238 (7%)

Query：20 ASHLAAFFQ-GFRSLEDIRSQA-SLTTQQAIGLKHYSDFLERMPREEATEIEQTVQKAAQ 77

ASH A ++Q G+R++E +R S T Q +GL+ Y DF + + EEATEI +T+

Sbjct：274 ASHAAEWYQKGWRTIEQVRKHKDSFTKQIKVGLEFYEDFCKTVTIEEATEIYETI---VS 330

Query：78 AFNSGLLCVAC--GSYRRGKATCGDVDVLITHPDGRSHRGIFSRLLDSLRQEGFLTDDLV 135

G+ +C G +RRGK DVD++++ S + + LL L +E V

Sbjct：331 RMPDGIKIQSCLVGGFRRGKPVGADVDMVLSPSHTHSTKHLVDVLLRILDEEFQFRLISV 390

Query：136 SQEENGQQQKYLGVC-XXXXXXXXXXXXDIIVVPYSEFACALLYFTGSAHFNRSMRALAK 194

+ G ++ Y+ + DIIVVP + A+L ++G F R ++ A

Sbjct：391 QEHSCGGKKGYVMLAVILSNSSKINRRVDIIVVPPAYIGSAVLGWSGGIFFLRDLKLYAN 450

Query：195 TK-GMSLSEHALSTAVVRNTHGCKVGPGRVL----PTPTEKDVFRLLGLPYREPAERD 247

+ G+S S ++ G + P P EKD+FR L Y EP R+

Sbjct：451 SHLGLSYD----SFEIINLKTGKDICPDEFNEWKDPVEAEKDIFRYFSLEYIEPKFRN 504

>PIR2：S58150 hypothetical protein SPAC2F7.06c-fission yeast

(Schizosaccharomyces pombe)

Length=506

Score value=80.8bits (196), predicated value=1e-14

Homogeny=70/238 (29%), similarity=110/238 (45%), breach=17/238 (7%)

Query：20 ASHLAAFFQ-GFRSLEDIRSQA-SLTTQQAIGLKHYSDFLERMPREEATEIEQTVQKAAQ 77

ASH A ++Q G+R++E +R S T Q +GL+ Y DF + + EEATEI +T+

Sbjct：274 ASHAAEWYQKGWRTIEQVRKHKDSFTKQIKVGLEFYEDFCKTVTIEEATEIYETI---VS 330

Query：78 AFNSGLLCVAC--GSYRRGKATCGDVDVLITHPDGRSHRGIFSRLLDSLRQEGFLTDDLV 135

G+ +C G +RRGK DVD++++ S + + LL L+E V

Sbjct：331 RMPDGIKIQSCLVGGFRRGKPVGADVDMVLSPSHTHSTKHLVDVLLRILDEEFQFRLISV 390

Query：136 SQEENGQQQKYLGVC-XXXXXXXXXXXXDIIVVPYSEFACALLYFTGSAHFNRSMRALAK 194

+ G ++ Y+ + DIIVVP + A+L ++G F R ++ A

Sbjct：391 QEHSCGGKKGYVMLAVILSNSSKINRRVDIIVVPPAYIGSAVLGWSGGIFFLRDLKLYAN 450

Query：195 TK-GMSLSEHALSTAVVRNTHGCKVGPGRVL----PTPTEKDVFRLLGLPYREPAERD 247

+ G+S S ++ G + P P EKD+FR L Y EP R+

Sbjct：451 SHLGLSYD----SFEIINLKTGKDICPDEFNEWKDPVEAEKDIFRYFSLEYIEPKFRN 504

3.PP1143

A: nucleotide sequence: (SEQ ID NO:7) length: 2060bp

1 CTCAGACAAG ATCTGCGTGG TCATCGACCT GGACGAGACC CTGGTGCACA

51 GCTCCTTCAA GCCAGTGAAC AACGCGGACT TCATCATCCC TGTGGAGATT

101 GATGGGGTGG TCCACCAGGT CTACGTGTTG AAGCGTCCTC ATGTGGATGA

151 GTTCCTGCAG CGAATGGGCG AGCTCTTTGA ATGTGTGCTG TTCACTGCTA

201 GCCTCGCCAA GTACGCAGAC CCAGTAGCTG ACCTGCTGGA CAAATGGGGG

251 GCCTTCCGGG CCCGGCTGTT TCGAGAGTCC TGCGTCTTCC ACCGGGGGAA

301 CTACGTGAAG GACCTGAGCC GGTTGGGTCG AGACCTGCGG CGGGTGCTCA

351 TCCTGGACAA TTCACCTGCC TCCTATGTCT TCCATCCAGA CAATGCTGTA

401 CCGGTGGCCT CGTGGTTTGA CAACATGAGT GACACAGAGC TCCACGACCT

451 CCTCCCCTTC TTCGAGCAAC TCAGCCGTGT GGACGACGTG TACTCAGTGC

501 TCAGGCAGCC ACGGCCAGGG AGCTAGTGAG GGTGATGGGG CCAGGACCTG

551 CCCCTGACCA ATGATACCCA CACCTCCTCC CAGGAAGACT GCCCAGGCCT

601 TTGTTAGGAA AACCCATGGG CCGCCGCCAC ACTCAGTGCC ATGGGGAAGC

651 GGGCGTCTCC CCCACCAGCC CCACCAGGCG GTGTAGGGGC AGCAGGCTGC

701 ACTGAGGACC GTGAGCTCCA GGCCCCGTGT CAGTGCCTTC AAACCTCCTC

751 CCCTATTCTC AGGGGACCTG GGGGGCCCTG CCTGCTGCTC CCTTTTTCTG

801 TCTCTGTCCA TGCTGCCATG TTTCTCTGCT GCCAAATTGG GCCCCTTGGC

851 CCCTTCCGGT TCTGCTTCCT GGGGGCAGGG TTCCTGCCTT GGACCCCCAG

901 TCTGGGAACG GTGGACATCA AGTGCCTTGC ATAGAGCCCC CTCTTCCCCG

951 CCCAGCTTTC CCAGGGGCAC AGCTCTAGGC TGGGAGGGGA GAACCAGCCC

1001 CTCCCCCTGC CCCACCTCCT CCCTTGGGAC TGAGAGGGCC CCTACCAACC

1051 TTTGCCTCTG CCTTGGAGGG AGGGGAGGTC TGTTACCACT GGGGAAGGCA

1101 GCAGGAGTCT GTCCTTCAGG CCCCACAGTG CAGCTTCTCC AGGGCCGACA

1151 GCTGAGGGCT GCTCCCTGCA TCATCCAAGC AATGACCTCA GACTTCTGCC

1201 TTAACCAGCC CCGGGGCTTG GCTCCCCCAG CTCTGAGCGT GGGGGCATAG

1251 GCAGGACCCC CCTTGTGGTG CCATATAAAT ATGTACATGT GTATATAGAT

1301 TTTTAGGGGA AGGAGAGAGG GAAGGGTCAG GGTAGAGACA CCCCTCCCTT

1351 GCCCCTTTCC TGGGCCCAGA AGTTGGGGGG AGGGAGGGAA AGGATTTTTA

1401 CATTTTTTAA ACTGCTATTT TCTGAATGGA ACAAGCTGGG CCAAGGGGCC

1451 CAGGCCCTGT CCTCTGTCCC TCACACCCCT TTGCTCCGTT CATTCATTCA

1501 AAAAAACATT TCTTGAGCAC CTTCTGTGCC CAGCATATGC TAGGCCCACC

1551 AGCTAAGTGT GTGTGGGGGG TCTCTACGCC AGCTCATCAG TGCCTCCTTG

1601 CCCATCCTTC ACCGGTGCCT TTGGGGGATC TGTAGGAGGT GGGACCTTCT

1651 GTGGGGTTTG GGGATCTCCA GGAAGCCCGA CCAAGCTGTC CCCTTCCCCT

1701 GTGCCAACCC ATCTCCTACA GCCCCCTGCC TGATCCCCTG CTGGCTGGGG

1751 GCAGCTCCCA GGATATCCTG CCTTCCAACT GTTTCTGAAG CCCCTCCTCC

1801 TAACATGGCG ATTCCGGAGG TCAAGGCCTT GGGCTCTCCC CAGGGTCTAA

1851 CGGTTAAGGG GACCCACATA CCAGTGCCAA GGGGGATGTC AAGTGGTGAT

1901 GTCGTTGTGC TCCCCTCCCC CAGAGCGGGT GGGCGGGGGG TGAATATGGT

1951 TGGCCTGCAT CAGGTGGCCT TCCCATTTAA GTGCCTTCTC TGTGACTGAG

2001 AGCCCTAGTG TGATGAGAAC TAAAGAGAAA GCCAGACCCC TAAAAAAAAA

2051 AAAAAAAAAA

B: aminoacid sequence: (SEQ ID NO:8) length: 146 amino acid

1 MLPCFSAAKL GPLAPSGSAS WGQGSCLGPP VWERWTSSAL HRAPSSPPSF

51 PRGTALGWEG RTSPSPCPTS SLGTERAPTN LCLCLGGRGG LLPLGKAAGV

101 CPSGPTVQLL QGRQLRAAPC IIQAMTSDFC LNQPRGLAPP ALSVGA

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

Clone number and protein name: PP1143

Start code: 810 ATG stop coding: 1250 TAG

Protein molecular weight: 14815.25

1 CT CAG ACA AGA TCT GCG TGG TCA TCG ACC TGG ACG AGA CCC TGG TGC 47

48 ACA GCT CCT TCA AGC CAG TGA ACA ACG CGG ACT TCA TCA TCC CTG TGG 95

96 AGA TTG ATG GGG TGG TCC ACC AGG TCT ACG TGT TGA AGC GTC CTC ATG 143

144 TGG ATG AGT TCC TGC AGC GAA TGG GCG AGC TCT TTG AAT GTG TGC TGT 191

192 TCA CTG CTA GCC TCG CCA AGT ACG CAG ACC CAG TAG CTG ACC TGC TGG 239

240 ACA AAT GGG GGG CCT TCC GGG CCC GGC TGT TTC GAG AGT CCT GCG TCT 287

288 TCC ACC GGG GGA ACT ACG TGA AGG ACC TGA GCC GGT TGG GTC GAG ACC 335

336 TGC GGC GGG TGC TCA TCC TGG ACA ATT CAC CTG CCT CCT ATG TCT TCC 383

384 ATC CAG ACA ATG CTG TAC CGG TGG CCT CGT GGT TTG ACA ACA TGA GTG 431

432 ACA CAG AGC TCC ACG ACC TCC TCC CCT TCT TCG AGC AAC TCA GCC GTG 479

480 TGG ACG ACG TGT ACT CAG TGC TCA GGC AGC CAC GGC CAG GGA GCT AGT 527

528 GAG GGT GAT GGG GCC AGG ACC TGC CCC TGA CCA ATG ATA CCC ACA CCT 575

576 CCT CCC AGG AAG ACT GCC CAG GCC TTT GTT AGG AAA ACC CAT GGG CCG 623

624 CCG CCA CAC TCA GTG CCA TGG GGA AGC GGG CGT CTC CCC CAC CAG CCC 671

672 CAC CAG GCG GTG TAG GGG CAG CAG GCT GCA CTG AGG ACC GTG AGC TCC 719

720 AGG CCC CGT GTC AGT GCC TTC AAA CCT CCT CCC CTA TTC TCA GGG GAC 767

768 CTG GGG GGC CCT GCC TGC TGC TCC CTT TTT CTG TCT CTG TCC ATG CTG 815

1 Met Leu 2

816 CCA TGT TTC TCT GCT GCC AAA TTG GGC CCC TTG GCC CCT TCC GGT TCT 863

3 Pro Cys Phe Ser Ala Ala Lys Leu Gly Pro Leu Ala Pro Ser Gly Ser 18

864 GCT TCC TGG GGG CAG GGT TCC TGC CTT GGA CCC CCA GTC TGG GAA CGG 911

19 Ala Ser Trp Gly Gln Gly Ser Cys Leu Gly Pro Pro Val Trp Glu Arg 34

912 TGG ACA TCA AGT GCC TTG CAT AGA GCC CCC TCT TCC CCG CCC AGC TTT 959

35 Trp Thr Ser Ser Ala Leu His Arg Ala Pro Ser Ser Pro Pro Ser Phe 50

960 CCC AGG GGC ACA GCT CTA GGC TGG GAG GGG AGA ACC AGC CCC TCC CCC 1007

51 Pro Arg Gly Thr Ala Leu Gly Trp Glu Gly Arg Thr Ser Pro Ser Pro 66

1008 TGC CCC ACC TCC TCC CTT GGG ACT GAG AGG GCC CCT ACC AAC CTT TGC 1055

67 Cys Pro Thr Ser Ser Leu Gly Thr Glu Arg Ala Pro Thr Asn Leu Cys 82

1056 CTC TGC CTT GGA GGG AGG GGA GGT CTG TTA CCA CTG GGG AAG GCA GCA 1103

83 Leu Cys Leu Gly Gly Arg Gly Gly Leu Leu Pro Leu Gly Lys Ala Ala 98

1104 GGA GTC TGT CCT TCA GGC CCC ACA GTG CAG CTT CTC CAG GGC CGA CAG 1151

99 Gly Val Cys Pro Ser Gly Pro Thr Val Gln Leu Leu Gln Gly Arg Gln 114

1152 CTG AGG GCT GCT CCC TGC ATC ATC CAA GCA ATG ACC TCA GAC TTC TGC 1199

115 Leu Arg Ala Ala Pro Cys Ile Ile Gln Ala Met Thr Ser Asp Phe Cys 130

1200 CTT AAC CAG CCC CGG GGC TTG GCT CCC CCA GCT CTG AGC GTG GGG GCA 1247

131 Leu Asn Gln Pro Arg Gly Leu Ala Pro Pro Ala Leu Ser Val Gly Ala 146

1248 TAG GCA GGA CCC CCC TTG TGG TGC CAT ATA AAT ATG TAC ATG TGT ATA 1295

147 *** 147

1296 TAG ATT TTT AGG GGA AGG AGA GAG GGA AGG GTC AGG GTA GAG ACA CCC 1343

1344 CTC CCT TGC CCC TTT CCT GGG CCC AGA AGT TGG GGG GAG GGA GGG AAA 1391

1392 GGA TTT TTA CAT TTT TTA AAC TGC TAT TTT CTG AAT GGA ACA AGC TGG 1439

1440 GCC AAG GGG CCC AGG CCC TGT CCT CTG TCC CTC ACA CCC CTT TGC TCC 1487

1488 GTT CAT TCA TTC AAA AAA ACA TTT CTT GAG CAC CTT CTG TGC CCA GCA 1535

1536 TAT GCT AGG CCC ACC AGC TAA GTG TGT GTG GGG GGT CTC TAC GCC AGC 1583

1584 TCA TCA GTG CCT CCT TGC CCA TCC TTC ACC GGT GCC TTT GGG GGA TCT 1631

1632 GTA GGA GGT GGG ACC TTC TGT GGG GTT TGG GGA TCT CCA GGA AGC CCG 1679

1680 ACC AAG CTG TCC CCT TCC CCT GTG CCA ACC CAT CTC CTA CAG CCC CCT 1727

1728 GCC TGA TCC CCT GCT GGC TGG GGG CAG CTC CCA GGA TAT CCT GCC TTC 1775

1776 CAA CTG TTT CTG AAG CCC CTC CTC CTA ACA TGG CGA TTC CGG AGG TCA 1823

1824 AGG CCT TGG GCT CTC CCC AGG GTC TAA CGG TTA AGG GGA CCC ACA TAG 1871

1872 CAG TGC CAA GGG GGA TGT CAA GTG GTG ATG TCG TTG TGC TCC CCT CCC 1919

1920 CCA GAG CGG GTG GGC GGG GGG TGA ATA TGG TTG GCC TGC ATC AGG TGG 1967

1968 CCT TCC CAT TTA AGT GCC TTC TCT GTG ACT GAG AGC CCT AGT GTG ATG 2015

2016 AGA ACT AAA GAG AAA GCC AGA CCC CTA AAA AAA AAA AAA AAA AAA 2060

D:Blastp result

Query=PP1143[gene=PP1143] (146 amino acid)

>SW：RPB1_CAEEL P16356 caenorhabditis elegans.dna-directed rna

polymerase ii largest subunit(ec 2.7.7.6).12/1998

Length=1859

Score value=30.9bits (68), predicated value=5.8

Homogeny=21/77 (27%), similarity=35/77 (45%), breach=1/77 (1%)

Query：3 PCFSAAKLGPLAPSGSASWGQGSCLGPPVWERWTSSALHRAPSSPPSFPRGTALGWEGRT 62

P LG L+P + G + P +++ ++ H +P+SP P A G +

Sbjct：1557 PASPGDPLGALSPRTPSYGGMSPGVYSPSSPQFSMTSPHYSPTSPSYSPTSPAAG-QSPV 1615

Query：63 SPSPCPTSSLGTERAPT 79

SPS PTS + +P+

Sbjct：1616 SPSYSPTSPSYSPTSPS 1632

Score value=30.5bits (67), predicated value=7.6

Homogeny=25/79 (31%), similarity=37/79 (46%), breach=6/79 (7%)

Query：6 SAAKLGPLAPSGSASWGQGSCLGP---PVWERWTSSALHRAPSSP---PSFPRGTALGWE 59

S+ K P +P+ S + S P P +++ S+ PSSP P+ PRG +

Sbjct：1726 SSPKYSPSSPTYSPTSPSYSPTSPQYSPTSPQYSPSSPTYTPSSPTYNPTSPRGFSSPQY 1785

Query：60 GRTSPSPCPTSSLGTERAP 78

TSP+ PTS T +P

Sbjct：1786 SPTSPTYSPTSPSYTPSSP 1804

>SP_IN：Q20090 Q20090 caenorhabditis elegans.c.elegans dna-directed

rna polymerase ii large subunit(ama-1)(sp：p16356).

11/1998

Length=1862

Score value=30.9bits (68), predicated value=5.8

Homogeny=21/77 (27%), similarity=35/77 (45%), breach=1/77 (1%)

Query：3 PCFSAAKLGPLAPSGSASWGQGSCLGPPVWERWTSSALHRAPSSPPSFPRGTALGWEGRT 62

P LG L+P + G + P +++ ++ H +P+SP P A G +

Sbjct：1560 PASPGDPLGALSPRTPSYGGMSPGVYSPSSPQFSMTSPHYSPTSPSYSPTSPAAG-QSPV 1618

Query：63 SPSPCPTSSLGTERAPT 79

SPS PTS + +P+

Sbjct：1619 SPSYSPTSPSYSPTSPS 1635

Score value=30.5bits (67), predicated value=7.6

Homogeny=25/79 (31%), similarity=37/79 (46%), breach=6/79 (7%)

Query：6 SAAKLGPLAPSGSASWGQGSCLGP---PVWERWTSSALHRAPSSP---PSFPRGTALGWE 59

S+ K P +P+ S + S P P +++ S+ PSSP P+ PRG +

Sbjct：1729 SSPKYSPSSPTYSPTSPSYSPTSPQYSPTSPQYSPSSPTYTPSSPTYNPTSPRGFSSPQY 1788

Query：60 GRTSPSPCPTSSLGTERAP 78

TSP+ PTS T +P

Sbjct：1789 SPTSPTYSPTSPSYTPSSP 1807

4.PP3241

A: nucleotide sequence (SEQ ID NO:10) length: 1682bp

1 GGAAAACAGA GGATAAAGAA GCCAAGTCTG GGAAGTTGGA AAAGGAGAAA

51 GAAGCAAAGG AAGGCTCTGA ACCAAAGGAG CAGGAAGACC TTCAAGAGAA

101 TGATGAGGAA GGCTCAGAAG ATGAAGCCTC GGAGACTGAC TACTCATCAG

151 CTGATGAGAA CATCCTCACC AAAGCAGATA CACTCAAAGT AAAGGATCGG

201 AAGAAGAAGA AGAAGAAAGG ACAGGAAGCA GGAGGATTTT TTTGAAGATG

251 CATCTCAGTA CGATGAAAAC CTCTCGTTCC AGGACATGAA CCTTTCCCGC

301 CCTCTTCTGA AGGCCATTAC AGCCATGGGC TTCAAGCAGC CCACCCCGAT

351 CCAGAAGGCG TGCATACCTG TGGGTCTATT GGGGAAGGAC ATCTGTGCCT

401 GTGCAGCCAC TGGGACAGGT AAAACTGCCG CCTTTGCCCT GCCTGTTTTG

451 GAGCGTCTGA TTTATAAACC CCGCCAGGCT CCAGTCACCC GCGTGCTGGT

501 GCTAGTGCCC ACCCGAGAGC TGGGCATCCA GGTGCACTCT GTCACCAGAC

551 AGCTGGCCCA GTTCTGCAAC ATCACCACCT GCCTGGCTGT GGGCGGCTTG

601 GATGTGAAGT CTCAGGAAGC AGCTCTTCGG GCAGCGCCTG ACATCCTCAT

651 CGCCACCCCA GGCCGGCTCA TCGATCACCT CCACAACTGC CCTTCCTTCC

701 ACCTGAGCAG CATCGAGGTG CTCATCCTGG ACGAGGCTGA CAGGATGCTG

751 GATGAGTACT TTGAGGAGCA GATGAAGGAG ATCATCCGAA TGTGTTCCCA

801 CCACCGCCAG ACCATGCTCT TCTCGGCCAC CATGACAGAC GAGGTGAAAG

851 ATCTGGCTTC TGTCTCCTTG AAGAATCCTG TCCGGATATT TGTGAACAAG

901 CAACACAGAT GTGGCTCCCT TCTGCGGCAG GAGTTCATCC GGATCCGGCC

951 TAATCGTGAA GGAGACCGGG AAGCCATCGT GGCAGCTTTG TTGACGAGGA

1001 CCTTCACTGA CCATGTGATG CTGTTCACGC AAACCAAGAA GCAGGCCCAC

1051 CGCATGCACA TCCTCCTGGG GCTCATGGGG CTGCAGGTGG GTGAGCTCCA

1101 TGGCAACTTG TCACAGACGC AGCGGCTGGA GGCCCTCCGG CGTTTTAAGG

1151 ATGAACAGAT TGACATCCTC GTGGCCACTG ATGTGGCAGC CCGTGGACTT

1201 GACATTGAGG GGTCAAAACG GTAATCAACT TCACAATGCC TAATACCATC

1251 AAACATTATG TCCACCGGGT GGGGCGAACA GCACGTGCTG GCAGGGCTGG

1301 GCGCTCAGTC TCTCTGGTGG GAGAAGATGA GCGGAAGATG CTGAAGGAGA

1351 TTGTAAAAGC TGCCAAGGCC CCTGTGAAGG CCAGGATACT TCCCCAAGAT

1401 GTCATCCTCA AATTCCGGGA CAAGATTGAG AAAATGGAGA AAGATGTGTA

1451 TGCAGTTCTG CAGCTAGAGG CGGAGGAAAA AGAGATGCAG CAGTCAGAAG

1501 CCCAGATCAA TACAGCAAAG CGGCTCCTGG AGAAGGGGAA GGAGGCAGTG

1551 GTCCAAGAGC CCGAGAGGAG CTGGTTCCAG ACCAAAGAAG AGAGGAAGAA

1601 GGAGAAAATT GCCAAAGCTC TGCAGGAATT TGACTTGGCC TTAAGAGGAA

1651 AGAAGAAAAG GAAGAAGTTT ATGAAGGATG CC

B: aminoacid sequence (SEQ ID NO:11) length: 312 amino acid

1 MNLSRPLLKA ITAMGFKQPT PIQKACIPVG LLGKDICACA ATGTGKTAAF

51 ALPVLERLIY KPRQAPVTRV LVLVPTRELG IQVHSVTRQL AQFCNITTCL

101 AVGGLDVKSQ EAALRAAPDI LIATPGRLID HLHNCPSFHL SSIEVLILDE

151 ADRMLDEYFE EQMKEIIRMC SHHRQTMLFS ATMTDEVKDL ASVSLKNPVR

201 IFVNKQHRCG SLLRQEFIRI RPNREGDREA IVAALLTRTF TDHVMLFTQT

251 KKQAHRMHIL LGLMGLQVGE LHGNLSQTQR LEALRRFKDE QIDILVATDV

301 AARGLDIEGS KR

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

Clone number and protein name: PP3241

Start code: 286 ATG stop coding: 1224 TAA

Protein molecular weight: 34808.97

1 GGA AAA CAG AGG ATA AAG AAG CCA AGT CTG GGA AGT TGG AAA AGG AGA 48

49 AAG AAG CAA AGG AAG GCT CTG AAC CAA AGG AGC AGG AAG ACC TTC AAG 96

97 AGA ATG ATG AGG AAG GCT CAG AAG ATG AAG CCT CGG AGA CTG ACT ACT 144

145 CAT CAG CTG ATG AGA ACA TCC TCA CCA AAG CAG ATA CAC TCA AAG TAA 192

193 AGG ATC GGA AGA AGA AGA AGA AGA AAG GAC AGG AAG CAG GAG GAT TTT 240

241 TTT GAA GAT GCA TCT CAG TAC GAT GAA AAC CTC TCG TTC CAG GAC ATG 288

1 Met 1

289 AAC CTT TCC CGC CCT CTT CTG AAG GCC ATT ACA GCC ATG GGC TTC AAG 336

2 Asn Leu Ser Arg Pro Leu Leu Lys Ala Ile Thr Ala Met Gly Phe Lys 17

337 CAG CCC ACC CCG ATC CAG AAG GCG TGC ATA CCT GTG GGT CTA TTG GGG 384

18 Gln Pro Thr Pro Ile Gln Lys Ala Cys Ile Pro Val Gly Leu Leu Gly 33

385 AAG GAC ATC TGT GCC TGT GCA GCC ACT GGG ACA GGT AAA ACT GCC GCC 432

34 Lys Asp Ile Cys Ala Cys Ala Ala Thr Gly Thr Gly Lys Thr Ala Ala 49

433 TTT GCC CTG CCT GTT TTG GAG CGT CTG ATT TAT AAA CCC CGC CAG GCT 480

50 Phe Ala Leu Pro Val Leu Glu Arg Leu Ile Tyr Lys Pro Arg Gln Ala 65

481 CCA GTC ACC CGC GTG CTG GTG CTA GTG CCC ACC CGA GAG CTG GGC ATC 528

66 Pro Val Thr Arg Val Leu Val Leu Val Pro Thr Arg Glu Leu Gly Ile 81

529 CAG GTG CAC TCT GTC ACC AGA CAG CTG GCC CAG TTC TGC AAC ATC ACC 576

82 Gln Val His Ser Val Thr Arg Gln Leu Ala Gln Phe Cys Asn Ile Thr 97

577 ACC TGC CTG GCT GTG GGC GGC TTG GAT GTG AAG TCT CAG GAA GCA GCT 624

98 Thr Cys Leu Ala Val Gly Gly Leu Asp Val Lys Ser Gln Glu Ala Ala 113

625 CTT CGG GCA GCG CCT GAC ATC CTC ATC GCC ACC CCA GGC CGG CTC ATC 672

114 Leu Arg Ala Ala Pro Asp Ile Leu Ile Ala Thr Pro Gly Arg Leu Ile 129

673 GAT CAC CTC CAC AAC TGC CCT TCC TTC CAC CTG AGC AGC ATC GAG GTG 720

130 Asp His Leu His Asn Cys Pro Ser Phe His Leu Ser Ser Ile Glu Val 145

721 CTC ATC CTG GAC GAG GCT GAC AGG ATG CTG GAT GAG TAC TTT GAG GAG 768

146 Leu Ile Leu Asp Glu Ala Asp Arg Met Leu Asp Glu Tyr Phe Glu Glu 161

769 CAG ATG AAG GAG ATC ATC CGA ATG TGT TCC CAC CAC CGC CAG ACC ATG 816

162 Gln Met Lys Glu Ile Ile Arg Met Cys Ser His His Arg Gln Thr Met 177

817 CTC TTC TCG GCC ACC ATG ACA GAC GAG GTG AAA GAT CTG GCT TCT GTC 864

178 Leu Phe Ser Ala Thr Met Thr Asp Glu Val Lys Asp Leu Ala Ser Val 193

865 TCC TTG AAG AAT CCT GTC CGG ATA TTT GTG AAC AAG CAA CAC AGA TGT 912

194 Ser Leu Lys Asn Pro Val Arg Ile Phe Val Asn Lys Gln His Arg Cys 209

913 GGC TCC CTT CTG CGG CAG GAG TTC ATC CGG ATC CGG CCT AAT CGT GAA 960

210 Gly Ser Leu Leu Arg Gln Glu Phe Ile Arg Ile Arg Pro Asn Arg Glu 225

961 GGA GAC CGG GAA GCC ATC GTG GCA GCT TTG TTG ACG AGG ACC TTC ACT 1008

226 Gly Asp Arg Glu Ala Ile Val Ala Ala Leu Leu Thr Arg Thr Phe Thr 241

1009 GAC CAT GTG ATG CTG TTC ACG CAA ACC AAG AAG CAG GCC CAC CGC ATG 1056

242 Asp His Val Met Leu Phe Thr Gln Thr Lys Lys Gln Ala His Arg Met 257

1057 CAC ATC CTC CTG GGG CTC ATG GGG CTG CAG GTG GGT GAG CTC CAT GGC 1104

258 His Ile Leu Leu Gly Leu Met Gly Leu Gln Val Gly Glu Leu His Gly 273

1105 AAC TTG TCA CAG ACG CAG CGG CTG GAG GCC CTC CGG CGT TTT AAG GAT 1152

274 Asn Leu Ser Gln Thr Gln Arg Leu Glu Ala Leu Arg Arg Phe Lys Asp 289

1153 GAA CAG ATT GAC ATC CTC GTG GCC ACT GAT GTG GCA GCC CGT GGA CTT 1200

290 Glu Gln Ile Asp Ile Leu Val Ala Thr Asp Val Ala Ala Arg Gly Leu 305

1201 GAC ATT GAG GGG TCA AAA CGG TAA TCA ACT TCA CAA TGC CTA ATA CCA 1248

306 Asp Ile Glu Gly Ser Lys Arg *** 313

1249 TCA AAC ATT ATG TCC ACC GGG TGG GGC GAA CAG CAC GTG CTG GCA GGG 1296

1297 CTG GGC GCT CAG TCT CTC TGG TGG GAG AAG ATG AGC GGA AGA TGC TGA 1344

1345 AGG AGA TTG TAA AAG CTG CCA AGG CCC CTG TGA AGG CCA GGA TAC TTC 1392

1393 CCC AAG ATG TCA TCC TCA AAT TCC GGG ACA AGA TTG AGA AAA TGG AGA 1440

1441 AAG ATG TGT ATG CAG TTC TGC AGC TAG AGG CGG AGG AAA AAG AGA TGC 1488

1489 AGC AGT CAG AAG CCC AGA TCA ATA CAG CAA AGC GGC TCC TGG AGA AGG 1536

1537 GGA AGG AGG CAG TGG TCC AAG AGC CCG AGA GGA GCT GGT TCC AGA CCA 1584

1585 AAG AAG AGA GGA AGA AGG AGA AAA TTG CCA AAG CTC TGC AGG AAT TTG 1632

1633 ACT TGG CCT TAA GAG GAA AGA AGA AAA GGA AGA AGT TTA TGA AGG ATG 1680

1681 CC 1682

E:Blastp result

Query=PP3241[gene=PP3241] (312 amino acid)

>SW：YAJ3_SCHPO Q09903 schizosaccharomyces pombe(fission yeast).

putative atp-dependent rna helicase c30d11.03.2/1996

Length=754

Score value=316bits (800), predicated value=2e-85

Homogeny=152/309 (49%), similarity=212/309 (68%)

Query：1 MNLSRPLLKAITAMGFKQPTPIQKACIPVGLLGKDIXXXXXXXXXXXXXXXLPVLERLIY 60

MNLSRP+LK ++ +GF+ PT IQ IP+ LLGKDI +P+LERL+Y

Sbjct：264 MNLSRPILKGLSNLGFEVPTQIQDKTIPLALLGKDIVGAAVTGSGKTAAFIVPILERLLY 323

Query：61 KPRQAPVTRVLVLVPTRELGIQVHSVTRQLAQFCNITTCLAVGGLDVKSQEAALRAAPDI 120

+P++ P TRVL+L PTREL +Q HSV ++A F +I CL +GGL +K QE LR PDI

Sbjct：324 RPKKVPTTRVLILCPTRELAMQCHSVATKIASFTDIMVCLCIGGLSLKLQEQELRKRPDI 383

Query：121 LIATPGRLIDHLHNCPSFHLSSIEVLILDEADRMLDEYFEEQMKEIIRMCSHHRQTMLFS 180

+IATPGR IDH+ N F + +IE++++DEADRML++ F +++ EII+ C RQTMLFS

Sbjct：384 VIATPGRFIDHMRNSQGFTVENIEIMVMDEADRMLEDGFADELNEIIQACPKSRQTMLFS 443

Query：181 ATMTDEVKDLASVSLKNPVRIFVNKQHRCGSLLRQEFIRIRPNREGDREAIVAALLTRTF 240

ATMTD+V DL +SL PVR+FV+ + LL QEF+R+RP RE R A++ L F

Sbjct：444 ATMTDKVDDLIRLSLNRPVRVFVDNKKTTAKLLTQEFVRVRPQRELLRPAMLIYLCKELF 503

Query：241 TDHVMLFTQTKKQAHRMHILLGLMGLQVGELHGNLSQTQRLEALRRFKDEQIDILVATDV 300

++F ++K AH+M ++ GL+ L E+HG+LSQ QR+AL F+D + + L+ATDV

Sbjct：504 HRRTIIFFRSKAFAHKMRVIFGLLSLNATEIHGSLSQEQRVRALEDFRDGKCNYLLATDV 563

Query：301 AARGLDIEG 309

A+RG+DI+G

Sbjct：564 ASRGIDIKG 572

>SW：DRS1_YEAST P32892 saccharomyces cerevisiae(baker’s yeast).

putative atp-dependent rna helicase drs1.11/1997

Length=752

Score value=296bits (749), predicated value=2e-79

Homogeny=144/311 (46%), similarity=223/311 (71%), breach=5/311 (1%)

Query：1 MNLSRPLLKAITAMGFKQPTPIQKACIPVGLLGKDIXXXXXXXXXXXXXXXLPVLERLIY 60

++LSRP+LK + ++G+ +P+PIQ A IP+ LLGKDI +P++ERL+Y

Sbjct：236 LSLSRPVLKGLASLGYVKPSPIQSATIPIALLGKDIIAGAVTGSGKTAAFMIPIIERLLY 295

Query：61 KPRQAPVTRVLVLVPTRELGIQVHSVTRQLAQFCN-ITTCLAVGGLDVKSQEAALRAAPD 119

KP + TRV+VL+PTREL IQV V +Q+A+F + IT LAVGGL+++ QE L++ PD

Sbjct：296 KPAKIASTRVIVLLPTRELAIQVADVGKQIARFVSGITFGLAVGGLNLRQQEQMLKSRPD 355

Query：120 ILIATPGRLIDHLHNCPSFHLSSIEVLILDEADRMLDEYFEEQMKEIIRMCSHHRQTMLF 179

I+IATPGR IDH+ N SF++ S+E+L++DEADRML+E F++++ EI+ + +RQ +LF

Sbjct：356 IVIATPGRFIDHIRNSASFNVDSVEILVMDEADRMLEEGFQDELNEIMGLLPSNRQNLLF 415

Query：180 SATMTDEVKDLASVSLKNPVRIFVNKQHRCGSLLRQEFIRIRPNREGDREAIVAALLTR- 238

SATM ++K L S+SLK PVRI ++ + + L QEF+RIR R+ + A++ L++

Sbjct：416 SATMNSKIKSLVSLSLKKPVRIMIDPPKKAATKLTQEFVRIR-KRDHLKPALLFNLIRKL 474

Query：239 --TFTDHVMLFTQTKKQAHRMHILLGLMGLQVGELHGNLSQTQRLEALRRFKDEQIDILV 296

T +++F K+ AHR+ I++GL+G+ VGELHG+L+Q QRL+++ +FK+ ++ +L+

Sbjct：475 DPTGQKRIVVFVARKETAHRLRIIMGLLGMSVGELHGSLTQEQRLDSVNKFKNLEVPVLI 534

Query：297 ATDVAARGLDI 307

TD+A+RGLDI

Sbjct：535 CTDLASRGLDI 545

5.PP3501

A: nucleotide sequence: (SEQ ID NO:13) length: 1796bp

1 CTGGGCTCGG GTGATCCTCC TGTCTTAGCC TTCCAAGGTG CTGGGATTAC

51 AGGTGTGGGC CACCATGCCC AGCCACCATC TTGGTTTAAT TTCTTTCCTT

101 GGAAATGGAG CAAAGGGCTT TCACTCTTCT TTTTCATGGC TGCATAGTAT

151 TCCATAATTG TTTAAACAAT TGATAATAGA CACTGTATCG GGGTGACTGG

201 TATACCCCCA AAATCACATC TGCCCAGAGC CTCAGGACAT GGCCTTGTTT

251 GGAAATGGTT TTAGCAGATG CAGCTAAGAT GAGGTCACCC CGTGTTAGGA

301 TGGCCCCGAA TCCAGTGCCT GGCACCCTTA TAAGGAGAGG GAAACTTGGA

351 CGCAGACAGG AAGGCCGGGT GAAGACAGAA GCAGAGACCG GGGGCCAAGG

401 AGTGCCTGGG GTTGCCCAGA GCCACCACGG GCTGCAAGAG GTGTGGGACA

451 GACTCTCTCA GCCACAGAAG GGCCCAGCCC CGCACAGGCC TGGATTTCAG

501 AGGGCGAACA TTTCTGTGGC CGAAGCTCCC AGTGCGTTTG CAGTCCTTGG

551 AGACGGCCGC AGACACATTT GTTCTTGGCC TGTCTCCAGG TCTGTAGCCC

601 CCACAGTGAG CGGCCACTGG CCGGAGGGGA ACAGGAAGAG CTGGAGGGGA

651 CGTCACACAC ATGAGCCTTT TAGGAATAAC GAGTGACACG TGCAAGACCC

701 CAAACCAAAC ACCATGTAAC CTGACACCCA GGGAGGGAGG GAGGGAGGGG

751 CTGGGAGGGG CTCACCTGTA GGGAGGGGCT CACCTGTACA GCGGTGCTGG

801 GAAGCTGGCC TCGAGGGTCC CCGAGGGGAT ACAGGGCCGT GCAGCCTCTT

851 GCCTGAGGGT TTGCATGTCT GCCTGGATCC CCCGGGCTCC TGCCAGAAGG

901 CCTCTGAGGC CACCGTTGAC AGCATAAAGG CCACCCAGTG TCTCGGCAAC

951 CACGCTGACC ACTCCCTCCT CCCAGCTGCG GGGTGAGGTG GCCTCGAGGG

1001 AAGGGCTGGA GAGTCGTTCT GAGTATTTAA CAACTGGGGG TGACACAGTG

1051 TGGGTGGGCA AGGCCAGGGC CACTGCCAGG TGGGGTCCGT GGCCCTGAGC

1101 CCCCAGCTCT ATGCACCCCC CACCTGGGGT CTGGCTTCTC CATCTCCACA

1151 CCCCCTTGAG GGGCTTCTGT CTCCCCCTGC CCCTTCGATC GCAGGAGGCA

1201 GTGCCTGGCC GGGGTCGCAG GCACCTGTCA CCCCAGCTCC TCACTCCTCA

1251 CCCACTCACA TCCAGTCCGT TTGTAAAATA CACCCAGGAT GAGACCTGCA

1301 CGCAAGTGGC TCACAGCAGC ACGATTTGTG ACAGCCCGAG GCGGAGAACA

1351 CCGAACACCC AGTGAAGGTG AGGGGATCAG CACGGCGCGG CCACCCACGC

1401 ACCCACGCGC TGGAATGAGA CTCAGCCACA AGGAGGTGCG AAGCTCTGAC

1451 CCAGGCCACA GTGCGGATGC ACCTTGAGGA TGTCACGCTC AGTGAGAGAC

1501 ACCAGACACA GAAGGGTACG CTGTGATCCC ACTTCTATGA AATGTCCAGG

1551 ACAGACCAAT CCACAGAATC AGGGAGAGGA TTCGTGGGTG CCGGGACTGG

1601 GGAGGGGGAC CTGGGGGTGA CTAGGTGACA TAATGGGGAC AGGGCTGCCT

1651 TCTGGGTGAT GAGAATGTTC TGGAATCAGA TGGGATGGCT GCACGGCGTG

1701 GTGAAGGTAC TGAACGCCAC CTCACTGTAA GACGGTAGAT TTTGTATTTT

1751 ACCACAATAA ACAAAACAAA ACAAAACCAA AAAAAAAAAA AAAAAA

B: aminoacid sequence: (SEQ ID NO:14) length: 143 amino acid

1 MVLADAAKMR SPRVRMAPNP VPGTLIRRGK LGRRQEGRVK TEAETGGQGV

51 PGVAQSHHGL QEVWDRLSQP QKGPAPHRPG FQRANISVAE APSAFAVLGD

101 GRRHICSWPV SRSVAPTVSG HWPEGNRKSW RGRHTHEPFR NNE

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

Clone number and protein name: PP3501

Start code: 255 ATG stop coding: 686 TGA

Protein molecular weight: 15739.00

1 CT GGG CTC GGG TGA TCC TCC TGT CTT AGC CTT CCA AGG TGC TGG GAT 47

48 TAC AGG TGT GGG CCA CCA TGC CCA GCC ACC ATC TTG GTT TAA TTT CTT 95

96 TCC TTG GAA ATG GAG CAA AGG GCT TTC ACT CTT CTT TTT CAT GGC TGC 143

144 ATA GTA TTC CAT AAT TGT TTA AAC AAT TGA TAA TAG ACA CTG TAT CGG 191

192 GGT GAC TGG TAT ACC CCC AAA ATC ACA TCT GCC CAG AGC CTC AGG ACA 239

240 TGG CCT TGT TTG GAA ATG GTT TTA GCA GAT GCA GCT AAG ATG AGG TCA 287

1 Met Val Leu Ala Asp Ala Ala Lys Met Arg Ser 11

288 CCC CGT GTT AGG ATG GCC CCG AAT CCA GTG CCT GGC ACC CTT ATA AGG 335

12 Pro Arg Val Arg Met Ala Pro Asn Pro Val Pro Gly Thr Leu Ile Arg 27

336 AGA GGG AAA CTT GGA CGC AGA CAG GAA GGC CGG GTG AAG ACA GAA GCA 383

28 Arg Gly Lys Leu Gly Arg Arg Gln Glu Gly Arg Val Lys Thr Glu Ala 43

384 GAG ACC GGG GGC CAA GGA GTG CCT GGG GTT GCC CAG AGC CAC CAC GGG 431

44 Glu Thr Gly Gly Gln Gly Val Pro Gly Val Ala Gln Ser His His Gly 59

432 CTG CAA GAG GTG TGG GAC AGA CTC TCT CAG CCA CAG AAG GGC CCA GCC 479

60 Leu Gln Glu Val Trp Asp Arg Leu Ser Gln Pro Gln Lys Gly Pro Ala 75

480 CCG CAC AGG CCT GGA TTT CAG AGG GCG AAC ATT TCT GTG GCC GAA GCT 527

76 Pro His Arg Pro Gly Phe Gln Arg Ala Asn Ile Ser Val Ala Glu Ala 91

528 CCC AGT GCG TTT GCA GTC CTT GGA GAC GGC CGC AGA CAC ATT TGT TCT 575

92 Pro Ser Ala Phe Ala Val Leu Gly Asp Gly Arg Arg His Ile Cys Ser 107

576 TGG CCT GTC TCC AGG TCT GTA GCC CCC ACA GTG AGC GGC CAC TGG CCG 623

108 Trp Pro Val Ser Arg Ser Val Ala Pro Thr Val Ser Gly His Trp Pro 123

624 GAG GGG AAC AGG AAG AGC TGG AGG GGA CGT CAC ACA CAT GAG CCT TTT 671

124 Glu Gly Asn Arg Lys Ser Trp Arg Gly Arg His Thr His Glu Pro Phe 139

672 AGG AAT AAC GAG TGA CAC GTG CAA GAC CCC AAA CCA AAC ACC ATG TAA 719

140 Arg Asn Asn Glu *** 144

720 CCT GAC ACC CAG GGA GGG AGG GAG GGA GGG GCT GGG AGG GGC TCA CCT 767

768 GTA GGG AGG GGC TCA CCT GTA CAG CGG TGC TGG GAA GCT GGC CTC GAG 815

816 GGT CCC CGA GGG GAT ACA GGG CCG TGC AGC CTC TTG CCT GAG GGT TTG 863

864 CAT GTC TGC CTG GAT CCC CCG GGC TCC TGC CAG AAG GCC TCT GAG GCC 911

912 ACC GTT GAC AGC ATA AAG GCC ACC CAG TGT CTC GGC AAC CAC GCT GAC 959

960 CAC TCC CTC CTC CCA GCT GCG GGG TGA GGT GGC CTC GAG GGA AGG GCT 1007

1008 GGA GAG TCG TTC TGA GTA TTT AAC AAC TGG GGG TGA CAC AGT GTG GGT 1055

1056 GGG CAA GGC CAG GGC CAC TGC CAG GTG GGG TCC GTG GCC CTG AGC CCC 1103

1104 CAG CTC TAT GCA CCC CCC ACC TGG GGT CTG GCT TCT CCA TCT CCA CAC 1151

1152 CCC CTT GAG GGG CTT CTG TCT CCC CCT GCC CCT TCG ATC GCA GGA GGC 1199

1200 AGT GCC TGG CCG GGG TCG CAG GCA CCT GTC ACC CCA GCT CCT CAC TCC 1247

1248 TCA CCC ACT CAC ATC CAG TCC GTT TGT AAA ATA CAC CCA GGA TGA GAC 1295

1296 CTG CAC GCA AGT GGC TCA CAG CAG CAC GAT TTG TGA CAG CCC GAG GCG 1343

1344 GAG AAC ACC GAA CAC CCA GTG AAG GTG AGG GGA TCA GCA CGG CGC GGC 1391

1392 CAC CCA CGC ACC CAC GCG CTG GAA TGA GAC TCA GCC ACA AGG AGG TGC 1439

1440 GAA GCT CTG ACC CAG GCC ACA GTG CGG ATG CAC CTT GAG GAT GTC ACG 1487

1488 CTC AGT GAG AGA CAC CAG ACA CAG AAG GGT ACG CTG TGA TCC CAC TTC 1535

1536 TAT GAA ATG TCC AGG ACA GAC CAA TCC ACA GAA TCA GGG AGA GGA TTC 1583

1584 GTG GGT GCC GGG ACT GGG GAG GGG GAC CTG GGG GTG ACT AGG TGA CAT 1631

1632 AAT GGG GAC AGG GCT GCC TTC TGG GTG ATG AGA ATG TTC TGG AAT CAG 1679

1680 ATG GGA TGG CTG CAC GGC GTG GTG AAG GTA CTG AAC GCC ACC TCA CTG 1727

1728 TAA GAC GGT AGA TTT TGT ATT TTA CCA CAA TAA ACA AAA CAA AAC AAA 1775

1776 ACC AAA AAA AAA AAA AAA AAA 1796

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 (5)

1. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) coding has the proteic polynucleotide of people of cancer suppressing function, and described albumen 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、SEQ ID NO：14；

(b) with polynucleotide (a) complementary polynucleotide.

2. polynucleotide as claimed in claim 1 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:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14.

3. polynucleotide as claimed in claim 1 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.

4. a carrier is characterized in that, it contains the described polynucleotide of claim 1.

5. 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 4;

(b) host cell that transforms or transduce with the described polynucleotide of claim 1.