CN1170843C - Noven huamn protein with function of promoting growth of cancer cell and its code sequence - Google Patents

Abstract

The present invention discloses a novel human protein with the function of promoting the growth of cancer cells, 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 promoting the growth of cancer cells.

Description

Have new people's albumen and the encoding sequence thereof that promote the growth of cancer cells function

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to new coding and have the proteic polynucleotide of people that promote the growth of cancer cells 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 development research people albumen and the agonist/inhibitor thereof relevant with growth of cancer cells.

Summary of the invention

The purpose of this invention is to provide people's protein polypeptide that new the having of a class promote the growth of cancer cells 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 promotion growth of cancer cells function is provided, it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5; 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 IDNO:5.

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 with the protein polypeptide that promotes the growth of cancer cells function that (a) coding is above-mentioned; (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, SEQID NO:5.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.

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

In a fourth aspect of the present invention, provide preparation to have the preparation method of the polypeptide of the protein-active that promotes the growth of cancer cells function, this method comprises: (a) being fit to express under the proteic condition with promotion growth of cancer cells function, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with the protein-active that promotes the growth of cancer cells function.

In a fifth aspect of the present invention, provide and the above-mentioned protein polypeptide specificity bonded antibody that promotes the growth of cancer cells function that has.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 promotion growth of cancer cells function of the present invention of safe and effective amount.These pharmaceutical compositions can be used for promoting the growth of cell.The present invention also provides a kind of pharmaceutical composition, it contain safe and effective amount at antibody and the pharmaceutically acceptable carrier with the protein polypeptide that promotes the growth of cancer cells function of the present invention.This pharmaceutical composition 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 that promotes the growth of cancer cells effect in acquisition, proves new gene through order-checking, further obtains full length cDNA clone.DNA transfection evidence, the albumen with promotion growth of cancer cells function of the present invention has the effect that promotes that the clone forms to cancer cells (liver cancer cell), and its promoter action 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 promotion growth of cancer cells function " is meant to have and promotes the protein polypeptide of growth of cancer cells function to be substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can have the albumen that promotes the growth of cancer cells 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 the protein polypeptide that promotes the growth of cancer cells 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 having the proteic fragment of people, derivative and the analogue that promotes the growth of cancer cells function.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep natural identical biological function or the active polypeptide of people's albumen that promotes the growth of cancer cells function that have 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 PP3999 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 IDNO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.Be example with PP4534 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 IDNO:6.Have the albumen that promotes the growth of cancer cells function for other,

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 that promotes the growth of cancer cells function to determine and/or to separate coding.

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.

Coding has the proteic specific DNA fragment sequence that promotes the growth of cancer cells function and 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, ColdSpring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) measure level with the proteic transcript that promotes the growth of cancer cells 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 protein product and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc. with the protein gene expression that promotes the growth of cancer cells 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 with carrier of the present invention or have the host cell that the albumen coded sequence that promotes the growth of cancer cells function produces through genetically engineered, 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 protein polypeptide (Science, 1984 that promote the growth of cancer cells function that have of reorganization; 224:1431).In general following steps are arranged:

(1). have the proteic polynucleotide of people (or varient) that promote the growth of cancer cells 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 promotion growth of cancer cells 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 contain and has people's encoding histone dna sequence dna of promoting the growth of cancer cells function and suitable transcribing/translate the expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

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

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

Having of reorganization promotes the people's albumen or the polypeptide of growth of cancer cells function to be 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 that promotes the growth of cancer cells function as pharmacological agent and be used to screen and promote or antagonism has antibody, polypeptide or other part of the protein function that promotes the growth of cancer cells function.For example, this antibody can be used for treating cancer or cellular abnormality propagation.Has the peptide molecule that can suppress or stimulate people's protein function that the people's protein screening peptide library that promotes the growth of cancer cells function can be used for seeking therapeutic value with the reorganization of expressing with promotion growth of cancer cells function.

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

Have the proteic antagonist of people that promotes the growth of cancer cells function and comprise antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.Have the proteic antagonist of people that promotes the growth of cancer cells function and can and eliminate its function with people's protein binding with promotion growth of cancer cells function, or suppress to have the proteic generation of people that promotes the growth of cancer cells function, or combine with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.Have and promote the proteic antagonist of people of growth of cancer cells function to can be used for therepic use.

In screening during as the compound of antagonist, can add in the bioanalysis mensuration having the albumen that promotes the growth of cancer cells function, the albumen and the interaction between its acceptor that have promotion growth of cancer cells function by the mensuration compounds affect determine 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.Have the albumen or its specific antibody that promote the growth of cancer cells function, can come administration by the amount that treats and/or prevents concrete indication effectively.Be applied to having of patient and promote the proteic amount and the dosage range of growth of cancer cells function will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Have and promote the proteic polynucleotide of people of growth of cancer cells function also to can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating since have that the proteic nothing that promotes the growth of cancer cells function is expressed or unusual/non-activity have cell development or a metabolic disturbance due to the proteic expression that promotes the growth of cancer cells function.The gene therapy vector (as virus vector) of reorganization can be designed to express the albumen that promotes the growth of cancer cells function that has of variation, to suppress the endogenic protein-active that promotes the growth of cancer cells function that has.For example, a kind of albumen that promotes the growth of cancer cells function that has of variation can be the albumen with promotion growth of cancer cells 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 and has the protein expression that promotes the growth of cancer cells function or the disease of active caused by abnormal.Deriving from the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for having and promotes the protein gene of growth of cancer cells function to be transferred in the cell.The method that structure carries the recombinant viral vector with the protein gene that promotes the growth of cancer cells function is found in existing document (Sambrook, et al.).Reorganization has the people's protein gene that promotes the growth of cancer cells function and can be packaged in the liposome and be transferred in the cell in addition.

Inhibition has the oligonucleotide (comprising sense-rna and DNA) of the people's protein mRNA that promotes the growth of cancer cells function 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.Because albumen of the present invention has the function that promotes growth of cancer cells, so the antisense sequences of albumen coded sequence of the present invention, can be introduced into cell to suppress the abnormality proliferation (as canceration) of cell.

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 at the antibody with the people's proteantigen determinant that promotes the growth of cancer cells 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 promotion growth of cancer cells function can be used in the immunohistochemistry technology, detects the people's albumen that promotes the growth of cancer cells function that has in the biopsy specimen.

The also available labelled with radioisotope of the protein bound monoclonal antibody of people with having promotion growth of cancer cells function injects 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 preventing and have the relevant disease of people's albumen of promotion growth of cancer cells function.The antibody that gives suitable dosage can stimulate or block and has proteic generation of people or the activity that promotes the growth of cancer cells function, thus the growth of anticancer and or the abnormality proliferation of cell.

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

Available people's albumen or the polypeptide immune animal of the production of polyclonal antibody with promotion growth of cancer cells 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 promote the growth of cancer cells function people's protein monoclonal antibody can with hybridoma technology production (Kohlerand 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.Pat No.4946778) also can be used for producing the anti-proteic single-chain antibody of people that promotes the growth of cancer cells function that has.

Can with have the protein bound peptide molecule of people that promotes the growth of cancer cells function and can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening and obtain.During screening, must promote people's protein molecular of growth of cancer cells function to carry out mark to having.

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

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

The sudden change that detection has the protein gene that promotes the growth of cancer cells function also can be used for diagnosing the relevant disease of albumen with promotion growth of cancer cells function.Form with the protein mutation that promotes the growth of cancer cells function comprises that to have point mutation that the protein D NA sequence that promotes the growth of cancer cells 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 Manualof Basic Techniques, 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 inMan (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 promotion growth of cancer cells 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 promotion growth of cancer cells 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 SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

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

PP3999 and PP4534 obtain 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 TMXRcDNA 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 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 hour, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2-3 time, there is the clone to form up to the microscopy cell, counting.Find that above 2 clones have the cell clone of promotion 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)
							PP3999	35	33	23	16	20	18
PP4534	26	33	48	23	21	14

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).

Embodiment 2: PCR 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.

The gene specific primer sequence

Clone's title	Special primer 1 (5 ' → 3 ')	Special primer 2 (5 ' → 3 ')
			PP3999	ACTTGGCCGGAAGGAAAGGTGCT	TGATGGAGATGGGATGCCTGTGA
PP4534	CAGGGGACACTCGGCAGCATCT	CTGCGCTCCTTCAGACTTGCTGC

Embodiment 3:cDNA cloned sequence is analyzed

1.PP3999

A: nucleotide sequence (SEQ ID NO:1) length: 1566bp

1 GGCAACCGAA AGGTTTCTGT AGTCAGAGCC CTTCGCCACG CCGTCCCGGC

51 AGGGATCCTC TGTTTAGGAC TGACGGTTGC TGTGGACTCT TATTTTTGGC

101 GGCAGCTCAC TTGGCCGGAA GGAAAGGTGC TTTGGTACAA CACTGTCCTG

151 AACAAAAGCT CCAACTGGGG GACCTCCCCG CTGCTGTGGT ACTTCTACTC

201 AGCCCTGCCC CGCGGCCTGG GCTGCAGCCT GCTCTTCATC CCCCTGGGCT

251 TGGTAGACAG AAGGACGCAC GCGCCGACGG TGCTGGCACT GGGCTTCATG

301 GCACTCTACT CCCTCCTGCC ACACAAGGAG CTACGCTTCA TCATCTATGC

351 CTTCCCCATG CTCAACATCA CGGCTGCCAG AGGCTGCTCC TACCTGCTGA

401 ATAACTATAA AAAGTCTTGG CTGTACAAAG CGGGGTCTCT GCTTGTGATC

451 GGACACCTCG TGGTGAATGC CGCCTACTCA GCCACGGCCC TGTATGTGTC

501 CCATTTCAAC TACCCAGGTG GCGTCGCAAT GCAGAGGCTG CACCAGCTGG

551 TGCCCCCCCA GACAGACGTC CTTCTGCACA TTGACGTGGC AGCCGCCCAG

601 ACAGGTGTGT CTCGGTTTCT CCAAGTCAAC AGCGCCTGGA GGTACGACAA

651 GAGGGAGGAT GTGCAGCCGG GGACAGGCAT GCTGGCATAC ACACACATCC

701 TCATGGAGGC GGCCCTGGGC TCCTGGCCCT CTACAGGGAC ACACACCGGG

751 TCCTGGCCAG CGTCGTGGGG ACCACAGGTG TGAGTCTGAA CCTGACCCAA

801 CTGCCCCCCT TCAACGTCCA CCTGCAGACA AAGCTGGTGC TTCTGGAGAG

851 GCTCCCCCGG CCGTCCTGAG GGGGACCAGG CAGCCCTCAG CAGCCACAGG

901 CCTTCCAGGA GCTGTTATCA CTACCAGTTT CTGGCACAAT TCCAGCACAA

951 TTATGACAAT TCAGAGAAGC AAGTCAAAGG ACTGGGCACC TGCCTCTGAC

1001 AGACACCAGA CCAGGTCCAG GGCCTCCTCC ACAGCCTCAG CTGGGGCTCT

1051 CAGCACCAAA GAACGAGGGG CCCAGGTCTT GTTGGCACCC CGGGAGCCAC

1101 TGCCCAGGGT GATGGTGGCC AGCTCAGGGC TTCCTGCGGG TGACTGTCGC

1151 CCAGACCAGG TGCCATTCAT GACTAATCAG GAGCAGCGGG CTCACCCAGG

1201 CACCTGTCTG CCAGGAGGCC ACGTGTGTCC TGCCCACCCA GGGGGAGCTG

1251 TATTTTGGCA GCACCCCACG CTTGCTGCCC GAGGGCCTCT TGGGGCACCT

1301 AAGACAGCAC CCCCTCTCAG GGGAGACCAT GGTGGCCCCG GCCGCACCCC

1351 CCCACCCTGG TGCCACCACT GCAACTTTTG TATTCACAGG CATCCCATCT

1401 CCATCACAGA TAAAATCTTA GGAGATAAAC ACATTCAAAA AGGAATGAGA

1451 TAAAAAGAAT AAGGCAATAA ATGTTGATTG GAACCTCTAA AAAAAAAAAA

1501 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1551 AAAAAAAAAA AAAAAA

B: aminoacid sequence (SEQ ID NO:2) length: 328 amino acid

1 MPPTQPRPCM CPISTTQVAS QCRGCTSWCP PRQTSFCTLT WQPPRQVCLG FSKSTAPGGT

61 TRGRMCSRGQ ACWHTHTSSW RRPWAPGPLQ GHTPGPGQRR GDHRCESEPD PTAPLQRPPA

121 DKAGASGEAP PAVLRGTRQP SAATGLPGAV ITTSFWHNSS TIMTIQRSKS KDWAPASDRH

181 QTRSRASSTA SAGALSTKER GAQVLLAPRE PLPRVMVASS GLPAGDCRPD QVPFMTNQEQ

241 RAHPGTCLPG GHVCPAHPGG AVFWQHPTLA ARGPLGAPKT APPLRGDHGG PGRTPPPWCH

301 HCNFCIHRHP ISITDKILGD KHIQKGMR

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

Clone number: PP3999

Start code: 467 ATG stop coding: 1453 TAA

Protein molecular weight: 35125

1 G GCA ACC GAA AGG TTT CTG TAG TCA GAG CCC TTC GCC ACG CCG TCC 46

47 CGG CAG GGA TCC TCT GTT TAG GAC TGA CGG TTG CTG TGG ACT CTT ATT 94

95 TTT GGC GGC AGC TCA CTT GGC CGG AAG GAA AGG TGC TTT GGT ACA ACA 142

143 CTG TCC TGA ACA AAA GCT CCA ACT GGG GGA CCT CCC CGC TGC TGT GGT 190

191 ACT TCT ACT CAG CCC TGC CCC GCG GCC TGG GCT GCA GCC TGC TCT TCA 238

239 TCC CCC TGG GCT TGG TAG ACA GAA GGA CGC ACG CGC CGA CGG TGC TGG 286

287 CAC TGG GCT TCA TGG CAC TCT ACT CCC TCC TGC CAC ACA AGG AGC TAC 334

335 GCT TCA TCA TCT ATG CCT TCC CCA TGC TCA ACA TCA CGG CTG CCA GAG 382

383 GCT GCT CCT ACC TGC TGA ATA ACT ATA AAA AGT CTT GGC TGT ACA AAG 430

431 CGG GGT GTC TGC TTG TGA TCG GAC ACC TCG TGG TGA ATG CCG CCT ACT 478

1 Met Pro Pro Thr 4

479 CAG CCA CGG CCC TGT ATG TGT CCC ATT TCA ACT ACC CAG GTG GCG TCG 526

5 Gln Pro Arg Pro Cys Met Cys Pro Ile Ser Thr Thr Gln Val Ala Ser 20

527 CAA TGCAGA GGC TGC ACC AGC TGG TGC CCC CCC AGA CAG ACG TCC TTC 574

21 Gln Cys Arg Gly Cys Thr Ser Trp Cys Pro Pro Arg Gln Thr Ser Phe 36

575 TGC ACA TTG ACG TGG CAG CCG CCC AGA CAG GTG TGT CTC GGT TTC TCC 622

37 Cys Thr Leu Thr Trp Gln Pro Pro Arg Gln Val Cys Leu Gly Phe Ser 52

623 AAG TCA ACA GCG CCT GGA GGT ACG ACA AGA GGG AGG ATG TGC AGC CGG 670

53 Lys Ser Thr Ala Pro Gly Gly Thr Thr Arg Gly Arg Met Cys Ser Arg 68

671 GGA CAG GCA TGC TGG CAT ACA CAC ACA TCC TCA TGG AGG CGG CCC TGG 718

69 Gly Gln Ala Cys Trp His Thr His Thr Ser Ser Trp Arg Arg Pro Trp 84

719 GCT CCT GGC CCT CTA CAG GGA CAC ACA CCG GGT CCT GGC CAG CGT CGT 766

85 Ala Pro Gly Pro Leu Gln Gly His Thr Pro Gly Pro Gly Gln Arg Arg 100

767 GGG GAC CAC AGG TGT GAG TCT GAA CCT GAC CCA ACT GCC CCC CTT CAA 814

101 Gly Asp His Arg Cys Glu Ser Glu Pro Asp Pro Thr Ala Pro Leu Gln 116

815 CGT CCA CCT GCA GAC AAA GCT GGT GCT TCT GGA GAG GCT CCC CCG GCC 862

117 Arg Pro Pro Ala Asp Lys Ala Gly Ala Ser Gly Glu Ala Pro Pro Ala 132

863 GTC CTG AGG GGG ACC AGG CAG CCC TCA GCA GCC ACA GGC CTT CCA GGA 910

133 Val Leu Arg Gly Thr Arg Gln Pro Ser Ala Ala Thr Gly Leu Pro Gly 148

911 GCT GTT ATC ACT ACC AGT TTC TGG CAC AAT TCC AGC ACA ATT ATG ACA 958

149 Ala Val Ile Thr Thr Ser Phe Trp His Asn Ser Ser Thr Ile Met Thr 164

959 ATT CAG AGA AGC AAG TCA AAG GAC TGG GCA CCT GCC TCT GAC AGA CAC 1006

165 Ile Gln Arg Ser Lys Ser Lys Asp Trp Ala Pro Ala Ser Asp Arg His 180

1007 CAG ACC AGG TCC AGG GCC TCC TCC ACA GCC TCA GCT GGG GCT CTC AGC 1054

181 Gln Thr Arg Ser Arg Ala Ser Ser Thr Ala Ser Ala Gly Ala Leu Ser 196

1055 ACC AAA GAA CGA GGG GCC CAG GTC TTG TTG GCA CCC CGG GAG CCA CTG 1102

197 Thr Lys Glu Arg Gly Ala Gln Val Leu Leu Ala Pro Arg Glu Pro Leu 212

1103 CCC AGG GTG ATG GTG GCC AGC TCA GGG CTT CCT GCG GGT GAC TGT CGC 1150

213 Pro Arg Val Met Val Ala Ser Ser Gly Leu Pro Ala Gly Asp Cys Arg 228

1151 CCA GAC CAG GTG CCA TTC ATG ACT AAT CAG GAG CAG CGG GCT CAC CCA 1198

229 Pro Asp Gln Val Pro Phe Met Thr Asn Gln Glu Gln Arg Ala His Pro 244

1199 GGC ACC TGT CTG CCA GGA GGC CAC GTG TGT CCT GCC CAC CCA GGG GGA 1246

245 Gly Thr Cys Leu Pro Gly Gly His Val Cys Pro Ala His Pro Gly Gly 260

1247 GCT GTA TTT TGG CAG CAC CCC ACG CTT GCT GCC CGA GGG CCT CTT GGG 1294

261 Ala Val Phe Trp Gln His Pro Thr Leu Ala Ala Arg Gly Pro Leu Gly 276

1295 GCA CCT AAG ACA GCA CCC CCT CTC AGG GGA GAC CAT GGT GGC CCC GGC 1342

277 Ala Pro Lys Thr Ala Pro Pro Leu Arg Gly Asp His Gly Gly Pro Gly 292

1343 CGC ACC CCC CCA CCC TGG TGC CAC CAC TGC AAC TTT TGT ATT CAC AGG 1390

293 Arg Thr Pro Pro Pro Trp Cys His His Cys Asn Phe Cys Ile His Arg 308

1391 CAT CCC ATC TCC ATC ACA GAT AAA ATC TTA GGA GAT AAA CAC ATT CAA 1438

309 His Pro Ile Ser Ile Thr Asp Lys Ile Leu Gly Asp Lys His Ile Gln 324

1439 AAA GGA ATG AGA TAA AAA GAA TAA GGC AAT AAA TGT TGA TTG GAA CCT 1486

325 Lys Gly Met Arg *** 329

1487 CTA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1534

1535 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA 1566

2.PP4534

A: nucleotide sequence (SEQ ID NO:4) length: 1683bp

1 AGGAATTTCA GCCAACATAA TAAGACATGA AAATGGCATT CGAGGTGTAT

51 TAGACAGACA AGGGGATGTT AGTGTTTGCA GGAGACTTGG TCTGCCTCAG

101 TGATGTCAGT CAGCAGTGAT TGTGATTCCC CAGGGGACAC TCGGCAGCAT

151 CTGGAGACAT TTTAGTTTAA ACTTCCCCAG TGATCTGTGA TGTACAGGAG

201 ACACTTTCGG TTGTCACACT GGGGGAGGAG GCTGCATGTC ACTGGCATCT

251 GTTGGGTGAC ACCTACAATG CACAGGACAA CCACAACAAA TAATTCAGGC

301 CCAAATGTTG CTGGTGCTGA GGGTGAGGTC CTAGTGTTAG TAACAGGAGG

351 AAAACCCAGC AGTCTGGAGG AGAGACCTCT TCCCAGGGCA GCCCAGGGGC

401 CATCAGGAGG GTTCATCTCA TGCATTAGAG GTCTTGGGAA GAATGAGGCT

451 TCCTTTCCTC CATCAAAGCA AGCAAATCCT TTAAAAGCTG CATCTCCAAG

501 GGCTGCTCCG GGCTCATAGC AAGCAACGTC GGAGCCCAGA GGCAAGGCTG

551 TGCTACTCAG CTGCCCTCTG GGGTCACAAA GGCTTCACTT GGCTTCTAAG

601 AGCTGATGAG GCCTCTCGCA AGGGACCCTG TGTGCATGGG CTGACCCTGA

651 AACTTCCCAG CCTCTCTTCT TCTCAGAGCA CCCTCAGGTG GCCTCTCGGG

701 GGTTACCCCT CATTGATACC ATGTCTCCTC GTGTTTTTGT CCAGACTCCA

751 ATTCCAGGGT TTCAGAACCG CATCGCAGCA TCTTTCCTGA AATGCACTCA

801 GACTCAGCCA GCAAAGACGT GCCTGGCCGC ATCCTGCTGG ATATAGACAA

851 TGATACCGAG AGCACTGCCC TGTGAAGAAA GCCCTTTCCC AGCCCTCCAC

901 CACTTCCACC CTGGCGAGTG GAGCAGGGGC AGGCGAACCT CTTTCTTTGC

951 AGACCGAACA GTGAAAAGCT TTCAGTGGAG GACAAAGGAG GGCCTCACTG

1001 TGCGGGACCT GGCCTTCTGC ACGGCCCAAG GAGAACCTGG AGGCCACCAC

1051 TAAAGCTGAA TGACCTGTGT CTTGAAGAAG TTGGCTTTCT TTACATGGGA

1101 AGGAAATCAT GCCAAAAAAA TCCAAAACAA AGAAGTACCT GGAGTGGAGA

1151 GAGTATTCCT GCTGAAACGC GCATAGGAAG CTTTTGTCCC TGCTGTTAAT

1201 GCGGGCAGCA CCTACAGCAA CTTGGAATGA GTAAGAAGCA GTGCGTTAAC

1251 TATCTATTTA ATAAAATGCG CTCATTATGC AAGTCGCCTA CTCTCTGCTA

1201 CCTGGACGTT CATTCTTATG TATTAGGAGG GAGGCTGCGC TCCTTCAGAC

1351 TTGCTGCAGA ATCATTTTGT ATCATGTATG GTCTGTGTCT CCCCAGTCCC

1401 CTCAGAACCA TGCCCATGGA TGGTGACTGC TGGCTCTGTC ACCTCATCAA

1451 ACTGGATGTG ACCCATGCCG CCTCGTTGGA TTGTCGGAAT GTAGACAGAA

1501 ATGTACTGTT CTTTTTTTTT TTTTTTAAAC AATGTAATTG CTACTTGATA

1551 AGGACCGAAC ATTATTCTAG TTTCATGTTT AATTTGAATT AAATATATTC

1601 TGTGGTTTGT GTGGAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1551 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA

B: aminoacid sequence (SEQ ID NO:5) length: 121 amino acid

1 MSKKQCVNYL FNKMRSLCKS PTLCYLDVHS YVLGGRLRSF RLAAESFCIM YGLCLPSPLR

61 TMPMDGDCWL CHLIKLDVTH AASLDCRNVD RNVLFFFFFF KQCNCYLIRT EHYSSFMFNL

121 N

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

Clone number: PP4534

Start code: 1227 ATG stop coding: 1592 TAA

Protein molecular weight: 14225

1 AG GAA TTT CAG CCA ACA TAA TAA GAC ATG AAA ATG GCA TTC GAG GTG 47

48 TAT TAG ACA GAC AAG GGG ATG TTA GTG TTT GCA GGA GAC TTG GTC TGC 95

96 CTC AGT GAT GTC AGT CAG CAG TGA TTG TGA TTC CCC AGG GGA CAC TCG 143

144 GCA GCA TCT GGA GAC ATT TTA GTT TAA ACT TCC CCA GTG ATC TGT GAT 191

192 GTA CAG GAG ACA CTT TCG GTT GTC ACA CTG GGG GAG GAG GCT GCA TGT 239

240 CAC TGG CAT CTG TTG GGT GAC ACC TAC AAT GCA CAG GAC AAC CAC AAC 287

288 AAA TAA TTC AGG CCC AAA TGT TGC TGG TGC TGA GGG TGA GGT CCT AGT 335

336 GTT AGT AAC AGG AGG AAA ACC CAG CAG TCT GGA GGA GAG ACC TCT TCC 383

384 CAG GGC AGC CCA GGG GCC ATC AGG AGG GTT CAT CTC ATG CAT TAG AGG 431

432 TCT TGG GAA GAA TGA GGC TTC CTT TCC TCC ATC AAA GCA AGC AAA TCC 479

480 TTT AAA AGC TGC ATC TCC AAG GGC TGC TCC GGG CTC ATA GCA AGC AAC 527

528 GTC GGA GCC CAG AGG CAA GGC TGT GCT ACT CAG CTG CCC TCT GGG GTC 575

576 ACA AAG GCT TCA CTT GGC TTC TAA GAG CTG ATG AGG CCT CTC GCA AGG 623

624 GAC CCT GTG TGC ATG GGC TGA CCC TGA AAC TTC CCA GCC TCT CTT CTT 671

672 CTC AGA GCA CCC TCA GGT GGC CTC TCG GGG GTT ACC CCT CAT TGA TAC 719

720 CAT GTC TCC TCG TGT TTT TGT CCA GAC TCC AAT TCC AGG GTT TCA GAA 767

768 CCG CAT CGC AGC ATC TTT CCT GAA ATG CAC TCA GAC TCA GCC AGC AAA 815

816 GAC GTG CCT GGC CGC ATC CTG CTG GAT ATA GAC AAT GAT ACC GAG AGC 863

864 ACT GCC CTG TGA AGA AAG CCC TTT CCC AGC CCT CCA CCA CTT CCA CCC 911

912 TGG CGA GTG GAG CAG GGG CAG GCG AAC CTC TTT CTT TGC AGA CCG AAC 959

960 AGT GAA AAG CTT TCA GTG GAG GAC AAA GGA GGG CCT CAC TGT GCG GGA 1007

1008 CCT GGC CTT CTG CAC GGC CCA AGG AGA ACC TGG AGG CCA CCA CTA AAG 1055

1056 CTG AAT GAC CTG TGT CTT GAA GAA GTT GGC TTT CTT TAC ATG GGA AGG 1103

1104 AAA TCA TGC CAA AAA AAT CCA AAA CAA AGA AGT ACC TGG AGT GGA GAG 1151

1152 AGT ATT CCT GCT GAA ACG CGC ATA GGA AGC TTT TGT CCC TGC TGT TAA 1199

1200 TGC GGG CAG CAC CTA CAG CAA CTT GGA ATG AGT AAG AAG CAG TGC GTT 1247

1 Met Ser Lys Lys Gln Cys Val 7

1248 AAC TAT CTA TTT AAT AAA ATG CGC TCA TTA TGC AAG TCG CCT ACT CTC 1295

8 Asn Tyr Leu Phe Asn Lys Met Arg Ser Leu Cys Lys Ser Pro Thr Leu 23

1296 TGC TAC CTG GAC GTT CAT TCT TAT GTA TTA GGA GGG AGG CTG CGC TCC 1343

24 Cys Tyr Leu Asp Val His Ser Tyr Val Leu Gly Gly Arg Leu Arg Ser 39

1344 TTC AGA CTT GCT GCA GAA TCA TTT TGT ATC ATG TAT GGT CTG TGT CTC 1391

40 Phe Arg Leu Ala Ala Glu Ser Phe Cys Ile Met Tyr Gly Leu Cys Leu 55

1392 CCC AGT CCC CTC AGA ACC ATG CCC ATG GAT GGT GAC TGC TGG CTC TGT 1439

56 Pro Ser Pro Leu Arg Thr Met Pro Met Asp Gly Asp Cys Trp Leu Cys 71

1440 CAC CTC ATC AAA CTG GAT GTG ACC CAT GCC GCC TCG TTG GAT TGT CGG 1487

72 His Leu Ile Lys Leu Asp Val Thr His Ala Ala Ser Leu Asp Cys Arg 87

1488 AAT GTA GAC AGA AAT GTA CTG TTC TTT TTT TTT TTT TTT AAA CAA TGT 1535

88 Asn Val Asp Arg Asn Val Leu Phe Phe Phe Phe Phe Phe Lys Gln Cys 103

1536 AAT TGC TAC TTG ATA AGG ACC GAA CAT TAT TCT AGT TTC ATG TTT AAT 1583

104 Asn Cys Tyr Leu Ile Arg Thr Glu His Tyr Ser Ser Phe Met Phe Asn 119

1584 TTG AAT TAA ATA TAT TCT GTG GTT TGT GTG GAA AAA AAA AAA AAA AAA 1631

120 Leu Asn *** 122

1632 AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1679

1680 AAA A 1683

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 (9)

1. isolating people's protein polypeptide with promotion growth of cancer cells function is characterized in that it is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5.

2. polypeptide as claimed in claim 1 is characterized in that, this amino acid sequence of polypeptide is selected from down group: SEQID NO:5.

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

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

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, the polypeptide of this polynucleotide encoding has the aminoacid sequence of the group of being selected from down: SEQ ID NO:2, SEQ ID NO:5.

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.

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

(a) being fit to express under the proteic condition of people with promotion growth of cancer cells function, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with the people's protein-active that promotes the growth of cancer cells function.

9. an energy and claim 1 are described has the people's protein polypeptide specificity bonded antibody that promotes the growth of cancer cells function.