CN1458169A - New human protein with function of improving mouse NIH/3T3 cell transformation and its encoding sequence - Google Patents
New human protein with function of improving mouse NIH/3T3 cell transformation and its encoding sequence Download PDFInfo
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- CN1458169A CN1458169A CN 02111645 CN02111645A CN1458169A CN 1458169 A CN1458169 A CN 1458169A CN 02111645 CN02111645 CN 02111645 CN 02111645 A CN02111645 A CN 02111645A CN 1458169 A CN1458169 A CN 1458169A
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Abstract
The present invention discloses a new kind of human protein with the functions of promoting 3T3 cell transformation, polynucleotides encoding this polypeptide and recombination process to produce the polypeptide. The present invention also discloses the agonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this new kind of human protein with the functions of promoting 3T3 cell transformation.
Description
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 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function is provided, it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:3,6,9; 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:3,6,9.
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 3T3 cell transformation 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:3,6,9.More preferably, the sequence of these polynucleotide is selected from down group: SEQ ID NO:2,5,8 coding region sequence or full length sequence.
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 3T3 cell transformation function, this method comprises: (a) being fit to express under the proteic condition with promotion 3T3 cell transformation function, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with the protein-active that promotes 3T3 cell transformation function.
In a fifth aspect of the present invention, provide and the above-mentioned protein polypeptide specificity bonded antibody that promotes 3T3 cell transformation function that has.The nucleic acid molecule that can be used for detecting also is provided, and it contains, and continuous 10 Nucleotide are to full length nucleotide in the above-mentioned polynucleotide, and preferably it contains the about 15-1000 of a successive Nucleotide.
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 3T3 cell transformation 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 antagonist (as antibody) and the pharmaceutically acceptable carrier with the protein polypeptide that promotes 3T3 cell transformation 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.
Embodiment
The 3T3 cell is a kind of l cell (J.Cell.Biol., 17:299,1963) (being also referred to as the NIH/3T3 cell).In the cancer research field, often foreign gene (especially people's gene) is introduced the 3T3 cell, observe its situation that influences to the growth of 3T3 cell.It has been generally acknowledged that, to 3T3 cell growth (or vicious transformation or transfection) influential gene is cancer related gene, wherein to 3T3 cell growth or transform that inhibiting gene is arranged is cancer suppressor gene mostly, and to the growth of 3T3 cell or transform (former) oncogene that has the gene of promoter action to be mostly.
The present invention adopts large-scale cDNA clone transfection mouse embryo fibroblasts 3T3, has on the basis that promotes the growth effect in acquisition, proves new gene through order-checking, further obtains full length cDNA clone.DNA transfection evidence, the albumen with promotion 3T3 cell transformation function of the present invention has the effect that promotes that the clone forms, its promotion rate 〉=50% to the 3T3 cell.
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 3T3 cell transformation function " is meant to have and promotes the protein polypeptide of 3T3 cell transformation 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 3T3 cell transformation function with the purified technology of protein purifying of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
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 3T3 cell transformation 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 3T3 cell transformation 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 PP14212 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:2 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that for PP14212 coding has the protein of SEQ ID NO:3, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:2.Be example with FP7030 albumen (in this application, its clone numbering is adopted in proteinic name) again, the coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:5 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that for FP7030 coding has the protein of SEQ ID NO:6, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQID NO:5.Other have the albumen that promotes 3T3 cell transformation function for the present invention, and the rest may be inferred.
The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:3 (is example with PP14212 albumen).
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 3T3 cell transformation 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 3T3 cell transformation 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.
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, ALaboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.
Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) function of marker gene occurs or forfeiture; (3) measure level with the proteic transcript that promotes 3T3 cell transformation 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 3T3 cell transformation function.
Use method (Saiki, the et al. Science1985 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 3T3 cell transformation function produces through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the protein polypeptide that promotes 3T3 cell transformation function that has of reorganization.In general following steps are arranged:
(1). have the proteic polynucleotide of people (or varient) that promote 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function and suitable transcribing/translate the expression vector of control signal.These methods comprise (Sambroook, et al) 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 that CMV is upright " early promoter, early stage and late period SV40 promotor and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.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 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function.For example, this antibody can be used for treating cancer or cellular abnormality propagation.The peptide molecule that can suppress or stimulate people's protein function that can be used for seeking therapeutic value with recombinant expressed protein screening peptide library of the present invention with promotion 3T3 cell transformation 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 3T3 cell transformation function to identify.Agonist improves and to have the people's albumen that promotes 3T3 cell transformation function biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.
Have the proteic antagonist of people that promotes 3T3 cell transformation function and comprise antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.Have the proteic antagonist of people that promotes 3T3 cell transformation function and can and eliminate its function with people's protein binding with promotion 3T3 cell transformation function, or suppress to have the proteic generation of people that promotes 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function, the albumen and the interaction between its acceptor that have promotion 3T3 cell transformation 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.
The proteic antagonist 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 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function is expressed or unusual/non-activity have cell development or a metabolic disturbance due to the proteic expression that promotes 3T3 cell transformation function.The gene therapy vector (as virus vector) of reorganization can be designed to express the albumen that promotes 3T3 cell transformation function that has of variation, to suppress the endogenic protein-active that promotes 3T3 cell transformation function that has.For example, a kind of albumen that promotes 3T3 cell transformation function that has of variation can be the albumen with promotion 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function to be transferred in the cell.The method that structure carries the recombinant viral vector with the protein gene that promotes 3T3 cell transformation function is found in existing document (Sambrook, et al.).Reorganization has the people's protein gene that promotes 3T3 cell transformation 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 3T3 cell transformation 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 the 3T3 cell transformation, 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.
The present invention also provides at the antibody with the people's proteantigen determinant that promotes 3T3 cell transformation 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 3T3 cell transformation function can be used in the immunohistochemistry technology, detects the people's albumen that promotes 3T3 cell transformation function that has in the biopsy specimen.
The also available labelled with radioisotope of the protein bound monoclonal antibody of people with having promotion 3T3 cell transformation 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 3T3 cell transformation function.The antibody that gives suitable dosage can be blocked proteic generation of people or the activity with promotion 3T3 cell transformation function, thus the abnormality proliferation of the growth of anticancer and/or 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 3T3 cell transformation 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 relevant positive cell (as cancer cells).
Available people's albumen or the polypeptide immune animal of the production of polyclonal antibody with promotion 3T3 cell transformation 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.
Having the people's protein monoclonal antibody that promotes 3T3 cell transformation function can be 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 3T3 cell transformation function that has.
Can with have the protein bound peptide molecule of people that promotes 3T3 cell transformation 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 3T3 cell transformation 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 3T3 cell transformation function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.That is detected in the test has a protein level that promotes 3T3 cell transformation function, can have the importance of albumen in various diseases that promotes 3T3 cell transformation function with laying down a definition and be used to diagnose to have the disease that the albumen that promotes 3T3 cell transformation function works.
Proteic polynucleotide with promotion 3T3 cell transformation function can be used for having the diagnosis and the treatment of the protein related diseases that promotes 3T3 cell transformation function.Aspect diagnosis, have the proteic polynucleotide that promotes 3T3 cell transformation function can be used for detecting have promote 3T3 cell transformation function proteic expression whether or under morbid state, have an abnormal exprssion that promotes 3T3 cell transformation function.As the protein D NA sequence with promotion 3T3 cell transformation function can be used for that the hybridization of biopsy specimen is had the proteic abnormal expression that promotes 3T3 cell transformation 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 gene chip), 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 3T3 cell transformation function with promotion 3T3 cell transformation function.
The sudden change that detection has the protein gene that promotes 3T3 cell transformation function also can be used for diagnosing the relevant disease of albumen with promotion 3T3 cell transformation function.Form with the protein mutation that promotes 3T3 cell transformation function comprises that to have point mutation that the protein D NA sequence that promotes 3T3 cell transformation 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).These sequences 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).Yet have only chromosomal marker thing seldom to can be used for the marker chromosomes position now based on actual sequence data (repetition polymorphism).For these sequences are associated with disease related gene.The first step is positioned dna sequence dna of the present invention 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.
Pyrenoids thuja acid full length sequence or its fragment with promotion 3T3 cell transformation 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 3T3 cell transformation 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.Notice that in Nucleotide and amino acid composite sequence, what (1) provided is the position that initial sum stops first Nucleotide of coding, (2) molecular weight unit is dalton.
The acquisition of embodiment 1:cDNA gene and the promoter action that mouse NIH/3T3 cell clone is formed
PP14212 obtains by making up the human placenta cDNA library with ordinary method; FP7030 and FP7915 come from the human fetal cDNA library that makes up with ordinary method.Get the placenta tissue (PP clone) or the fetal tissue (FP clone) 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 anticancer growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen96 orifice plate plasmid extraction test kit, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously mouse NIH/3T3 cell.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 the mouse NIH/3T3 cell 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-mentioned clone has the cell clone of promotion formation effect, the result is as shown in the table.
CDNA clone's transfectional cell (3T3) clone formation situation
| CDNA clones title | CDNA clones number (three repetitions) | Empty carrier clone number (three repetitions) | ||||
| PP14212 | ????40 | ????51 | ????48 | ????38 | ????37 | ????35 |
| FP7030 | ????58 | ????60 | ????67 | ????21 | ????28 | ????20 |
| FP7915 | ????60 | ????65 | ????69 | ????12 | ????18 | ????17 |
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).
Embodiment 2: PCR obtains full-length gene from placenta or fetus cDNA:
Get the placenta tissue (PP clone) or the fetal tissue (FP clone) at 3,6,9 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.With MMLV-RT-Superscript II (GIBCO BRL), ThermoScript II is carried out reverse transcription reaction at 42 ℃, obtains placenta or fetus cDNA.Utilize the special primer (as shown in the table) of each gene, by 97 ℃ of 3 ' 1 circulations.94 ℃ 30 " 60 ℃ 30 " 72 ℃ of 1 ' 35 circulations, pcr amplification is carried out in 72 ℃ of 10 ' 1 circulations, and acquisition contains the amplified production of each protein gene of complete open reading frame sequence.Amplified production is through sequence verification, and the sequence that records with embodiment 1 conforms to, and changes amplified production over to host cell with routine techniques subsequently, obtains recombinant protein (SEQ ID NO:3,6,9).
Gene specific primer
| Clone's title | Special primer 1 (5 ' → 3 ') | ????SEQ?ID ????NO： | Special primer 2 (3 ' → 5 ') | ????SEQ?ID ????NO： |
| ??PP14212 | ??(44)tgtgaacaccttgagccttg | ????10 | (1555)tgcaacctctgcctcaca | ????11 |
| ??FP7030 | ??(79)tcagcgtgtgcagtctctct | ????12 | (3587)tttttgagtggccaaagtcc | ????13 |
| ??FP7915 | ??(3)gcactgtgtgtttggacagc | ????14 | (3396)tatccttccgccacaaaaac | ????15 |
Annotate: in the bracket is the correspondence position of primer in each gene DNA sequence.
Embodiment 3:cDNA cloned sequence is analyzed
1.PP14212 albumen
A: nucleotide sequence (SEQ ID NO:1) length: 1637 bases
B: aminoacid sequence (SEQ ID NO:3) length: 288 amino acid
C. Nucleotide and amino acid composite sequence (SEQ ID NO:2) clone number and protein name: PP14212
Start code: 64 ATG stop coding: 928 TAG protein molecular weight: 31640.56Da
2.FP7030 albumen
A: nucleotide sequence (SEQ ID NO:4) length: 3600 bases
B: aminoacid sequence (SEQ ID NO:6) length: 307 amino acid
C. Nucleotide and amino acid composite sequence (SEQ ID NO:5) clone number and protein name: FP7030
Start code: 585 ATG stop coding: 1506 TGA protein molecular weight: 32581.89Da
3.FP7915 albumen
A: nucleotide sequence (SEQ ID NO:7) length: 3491 bases
B: aminoacid sequence (SEQ ID NO:9) length: 178 amino acid
C. Nucleotide and amino acid composite sequence (SEQ ID NO:8) clone number and protein name: FP7915
Start code: 336 ATG stop coding: 870 TGA protein molecular weight: 19443.65Da
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.
＜110〉 ＜120〉 NIH/3T3＜130〉 022537＜160〉 15＜170〉 PatentIn version 3.0＜210〉 1＜211〉 1637＜212〉 DNA＜213〉 ( Homo sapiens )＜400〉 1gggctttcaa cccccacctc agcccagcaa ttcgtttgga gcatgtgaac accttgagcc 60ttgatgagtt ccagtatgtg gtatattatg cagagcattc agagcaaata ctctctctcc 120gagcgcttaa tccgaacaat tgctgccatc cgttccttcc cacatgataa tgtagaggac 180ctcatcagag ggggagcaga tgtgaactgc actcatggca cactgaagcc cttgcactgt 240gcctgtatgg tgtcagatgc tgactgtgtg gagttacttc tggaaaaagg agccgaggtg 300aatgccctgg atgggtataa ccgaacagcc ctccactatg cagcagagaa agatgaggct 360tgtgtggagg tcctattgga gtatggtgca aaccccaatg ctttggatgg caacagagat 420accccacttc actgggcagc ctttaagaac aatgctgagt gtgtgcgggc tctcctagag 480agcggggcct ctgtcaatgc cctggattac aacaatgata caccgctcag ctgggctgcc 540atgaagggaa atcttgagag tgtcagcatc cttctggatt atggcgcaga ggtcagagtc 600atcaacctaa taggccagac acccatctcc cgcctggtgg ctctgctagt caggggactt 660ggaacagaga aagaggactc ttgctttgag ctcctccaca gagctgttgg acactttgaa 720ttgaggaaaa atggcaccat gccacgagag gtggccagag acccgcagct atgtgaaaaa 780ctgactgttc tgtgctcagc tccaggaact ctaaaaacac tcgctcgcta tgccgtgcgc 840cgtagcctgg gactccagta tctccccgat gcagtgaagg gccttccact gccagcttct 900ttgaaggaat acctgttact tttagaatag ccggagaaga tgtttgcacc atcgtgcagg 960cagctctggg tgaggttgtc cctgcagtac tccttgtcac agaaaacaga aaaacagttg 1020ttcctgttgt gtggtttata gatttcgaag caacatgtca caacaataac ctccatagca 1080cctccccttc ccaaaccaaa caacccaaca aaaaaaatcc ctcacttttg ttttctgttt 1140attgcttacc tggcttttta tattgcattt tgcaaaagaa gaggtctccc tcaatcctcc 1200cctttaggga aggagtcaac agtgtaacta aatttctcta ggaagatgga aagtacttaa 1260ataatgtgtg tgtggttttc tttggggacg tggttaacgg tccagaagaa tcccttctag 1320aaagcatttt aggccagcca tggtggctca cgtctgtaat cccaggactt tgggaggctg 1380aggcaggtgg atcacctgag gtcaggagtt cgagcccagc ctgaccaata tgatgaaacc 1440ccgtctctac taaaaataca aaaattagct gggcatggtg gcatgcgcct gtaatcccag 1500ctactcagga ggctgagaca gaagaatcgc ttgaacctgt gaggcagagg ttgcagtgag 1560ccaagatcgc gccattgcac tccagcctgg acaacaagag caaaactgtc tcaaaaaaaa 1620aaaaaaaaaa aaaaaaa 1637＜210〉2＜211〉1637＜212〉DNA＜213〉 ( Homo sapiens )＜220〉＜221〉CDS＜222〉 ( 64 ) .. ( 927 )＜400〉2gggctttcaa cccccacctc agcccagcaa ttcgtttgga gcatgtgaac accttgagcc 60ttg atg agt tcc agt atg tgg tat att atg cag agc att cag agc aaa 108
Met?Ser?Ser?Ser?Met?Trp?Tyr?Ile?Met?Gln?Ser?Ile?Gln?Ser?Lys
1???????????????5???????????????????10??????????????????15tac?tct?ctc?tcc?gag?cgc?tta?atc?cga?aca?att?gct?gcc?arc?cgt?tcc??????156Tyr?Ser?Leu?Ser?Glu?Arg?Leu?Ile?Arg?Thr?Ile?Ala?Ala?Ile?Arg?Ser
20??????????????????25??????????????????30ttc?cca?cat?gat?aat?gta?gag?gac?crc?atc?aga?ggg?gga?gca?gat?gtg??????204Phe?Pro?His?Asp?Asn?Val?Glu?Asp?Leu?Ile?Arg?Gly?Gly?Ala?Asp?Val
35??????????????????40??????????????????45aac?tgc?act?cat?ggc?aca?ctg?aag?ccc?ttg?cac?tgt?gcc?tgt?atg?gtg??????252Asn?Cys?Thr?His?Gly?Thr?Leu?Lys?Pro?Leu?His?Cys?Ala?Cys?Met?Val
50??????????????????55??????????????????60rca?gat?gct?gac?tgt?gtg?gag?tta?ctt?ctg?gaa?aaa?gga?gcc?gag?gtg??????300Set?Asp?Ala?Asp?Cys?Val?Glu?Leu?Leu?Leu?Glu?Lys?Gly?Ala?Glu?Val
65??????????????????70??????????????????75aat?gcc?ctg?gat?ggg?tat?aac?cga?aca?gcc?ctc?cac?tat?gca?gca?gag??????348Asn?Ala?Leu?Asp?Gly?Tyr?Asn?Arg?Thr?Ala?Leu?His?Tyr?Ala?Ala?Glu80??????????????????85??????????????????90??????????????????95aaa?gat?gag?gct?tgt?gtg?gag?gtc?cta?ttg?gag?tat?ggt?gca?aac?ccc??????396Lys?Asp?Glu?Ala?Cys?Val?Glu?Val?Leu?Leu?Glu?Tyr?Gly?Ala?Asn?Pro
100?????????????????105?????????????????110aat?gct?ttg?gat?ggc?aac?aga?gat?acc?cca?ctt?cac?tgg?gca?gcc?ttt??????444Asn?Ala?Leu?Asp?Gly?Asn?Arg?Asp?Thr?Pro?Leu?His?Trp?Ala?Ala?Phe
115?????????????????120?????????????????125aag?aac?aat?gct?gag?tgt?gtg?cgg?gct?ctc?cta?gag?agc?ggg?gcc?tct??????492Lys?Asn?Asn?Ala?Glu?Cys?Val?Arg?Ala?Leu?Leu?Glu?Ser?Gly?Ala?Ser
130?????????????????135?????????????????140gtc?aat?gcc?ctg?gat?tac?aac?aat?gat?aca?ccg?ctc?agc?tgg?gct?gcc??????540Val?Asn?Ala?Leu?Asp?Tyr?Asn?Asn?Asp?Thr?Pro?Leu?Ser?Trp?Ala?Ala
145?????????????????150?????????????????155atg?aag?gga?aat?crt?gag?agt?gtc?agc?atc?ctt?ctg?gat?tat?ggc?gca??????588Met?Lys?Gly?Asn?Leu?Glu?Ser?Val?Ser?Ile?Leu?Leu?Asp?Tyr?Gly?Ala160?????????????????165?????????????????170?????????????????175gag?gtc?aga?gtc?arc?aac?cta?ata?ggc?cagaca?ccc?atc?tcc?cgc?ctg???????636Glu?Val?Arg?Val?Ile?Asn?Leu?Ile?Gly?Gln?Thr?Pro?Ile?Ser?Arg?Leu
180?????????????????185?????????????????190gtg?gct?ctg?cta?gtc?agg?gga?ctt?gga?aca?gag?aaa?gag?gac?tct?tgc??????684Val?Ala?Leu?Leu?Val?Arg?Gly?Leu?Gly?Thr?Glu?Lys?Glu?Asp?Ser?Cys
195?????????????????200?????????????????205ttt?gag?ctc?ctc?cac?aga?gct?gtt?gga?cac?ttt?gaa?ttg?agg?aaa?aat??????732Phe?Glu?Leu?Leu?His?Arg?Ala?Val?Gly?His?Phe?Glu?Leu?Arg?Lys?Asn
210?????????????????215?????????????????220ggc?acc?atg?cca?cga?gag?gtg?gcc?aga?gac?ccg?cag?cta?tgt?gaa?aaa??????780Gly?Thr?Met?Pro?Arg?Glu?Val?Ala?Arg?Asp?Pro?Gln?Leu?Cys?Glu?Lys
225?????????????????230?????????????????235ctg?act?gtt?ctg?tgc?tca?gct?cca?gga?act?cta?aaa?aca?ctc?gct?cgc??????828Leu?Thr?Val?Leu?Cys?Ser?Ala?Pro?Gly?Thr?Leu?Lys?Thr?Leu?Ala?Arg240?????????????????245?????????????????250?????????????????255tat?gcc?gtg?cgc?cgt?agc?ctg?gga?ctc?cag?tat?ctc?ccc?gat?gca?gtg??????876Tyr?Ala?Val?Arg?Arg?Ser?Leu?Gly?Leu?Gln?Tyr?Leu?Pro?Asp?Ala?Val
260?????????????????265?????????????????270aag?ggc?ctt?cca?ctg?cca?gct?tct?ttg?aag?gaa?tac?ctg?tta?ctt?tta??????924Lys?Gly?Leu?Pro?Leu?Pro?Ala?Ser?Leu?Lys?Glu?Tyr?Leu?Leu?Leu?Leu
275 280 285gaa tagccggaga agatgtttgc accatcgtgc aggcagctct gggtgaggtt 977Glugtccctgcag tactccttgt cacagaaaac agaaaaacag ttgttcctgt tgtgtggttt 1037atagatttcg aagcaacatg tcacaacaat aacctccata gcacctcccc ttcccaaacc 1097aaacaaccca acaaaaaaaa tccctcactt ttgttttctg tttattgctt acctggcttt 1157ttatattgca ttttgcaaaa gaagaggtct ccctcaatcc tcccctttag ggaaggagtc 1217aacagtgtaa ctaaatttct ctaggaagat ggaaagtact taaataatgt gtgtgtggtt 1277ttctttgggg acgtggttaa cggtccagaa gaatcccttc tagaaagcat tttaggccag 1337ccatggtggc tcacgtctgt aatcccagga ctttgggagg ctgaggcagg tggatcacct 1397gaggtcagga gttcgagccc agcctgacca atatgatgaa accccgtctc tactaaaaat 1457acaaaaatta gctgggcatg gtggcatgcg cctgtaatcc cagctactca ggaggctgag 1517acagaagaat cgcttgaacc tgtgaggcag aggttgcagt gagccaagat cgcgccattg 1577cactccagcc tggacaacaa gagcaaaact gtctcaaaaa aaaaaaaaaa aaaaaaaaaa 1637＜210〉3＜211〉288＜212〉PRT＜213〉 ( Homo sapiens )＜400〉3Met Ser Ser Ser Met Trp Tyr Ile Met Gln Ser Ile Gln Ser Lys Tyr1 5 10 15Ser Leu Ser Glu Arg Leu Ile Arg Thr Ile Ala Ala Ile Arg Ser Phe
20??????????????????25??????????????????30Pro?His?Asp?Asn?Val?Glu?Asp?Leu?Ile?Arg?Gly?Gly?Ala?Asp?Val?Asn
35??????????????????40??????????????????45Cys?Thr?His?Gly?Thr?Leu?Lys?Pro?Leu?His?Cys?Ala?Cys?Met?Val?Ser
50??????????????????55??????????????????60Asp?Ala?Asp?Cys?Val?Glu?Leu?Leu?Leu?Glu?Lys?Gly?Ala?Glu?Val?Asn65??????????????????70??????????????????75??????????????????80Ala?Leu?Asp?Gly?Tyr?Asn?Arg?Thr?Ala?Leu?His?Tyr?Ala?Ala?Glu?Lys
85??????????????????90??????????????????95Asp?Glu?Ala?Cys?Val?Glu?Val?Leu?Leu?Glu?Tyr?Gly?Ala?Asn?Pro?Asn
100?????????????????105?????????????????110Ala?Leu?Asp?Gly?Asn?Arg?Asp?Thr?Pro?Leu?His?Trp?Ala?Ala?Phe?Lys
115?????????????????120?????????????????125Asn?Asn?Ala?Glu?Cys?Val?Arg?Ala?Leu?Leu?Glu?Ser?Gly?Ala?Ser?Val
130?????????????????135?????????????????140Asn?Ala?Leu?Asp?Tyr?Asn?Asn?Asp?Thr?Pro?Leu?Ser?Trp?Ala?Ala?Met145?????????????????150?????????????????155?????????????????160Lys?Gly?Asn?Leu?Glu?Ser?Val?Ser?Ile?Leu?Leu?Asp?Tyr?Gly?Ala?Glu
165?????????????????170?????????????????175Val?Arg?Val?Ile?Asn?Leu?Ile?Gly?Gln?Thr?Pro?Ile?Ser?Arg?Leu?Val
180?????????????????185?????????????????190Ala?Leu?Leu?Val?Arg?Gly?Leu?Gly?Thr?Glu?Lys?Glu?Asp?Ser?Cys?Phe
195?????????????????200?????????????????205Glu?Leu?Leu?His?Arg?Ala?Val?Gly?His?Phe?Glu?Leu?Arg?Lys?Asn?Gly
210?????????????????215?????????????????220Thr?Met?Pro?Arg?Glu?Val?Ala?Arg?Asp?Pro?Gln?Leu?Cys?Glu?Lys?Leu225?????????????????230?????????????????235?????????????????240Thr?Val?Leu?Cys?Ser?Ala?Pro?Gly?Thr?Leu?Lys?Thr?Leu?Ala?Arg?Tyr
245?????????????????250?????????????????255Ala?Val?Arg?Arg?Ser?Leu?Gly?Leu?Gln?Tyr?Leu?Pro?Asp?Ala?Val?Lys
260?????????????????265?????????????????270Gly?Leu?Pro?Leu?Pro?Ala?Ser?Leu?Lys?Glu?Tyr?Leu?Leu?Leu?Leu?Glu
275 280 285＜210〉4＜211〉3600＜212〉DNA＜213〉 (Homo sapiens)＜400〉4gctttcttct ctctctgtta tctctcttgt cgtctttttg tccttatctt ggtctctctg 60cttctatttc tacctctctc agcgtgtgca gtctctcttt ccctgtatgt ctactgtctt 120tctccgagtc tctcttcttc tccccgcccc cctcactccc ctttctccgt ccctctctgt 180ctctgcgcct ttctcgccct cactttctcc cctcctgcct gctccgggcc tgcccccacc 240gcgcgccggt tcaggggaac gtgtgcggag cgattgtcct gggggacatt tgatggatta 300gagcgccgag cggttccatt gctaggggac cacctgatct ccagcctgcg tcccatataa 360agccggcagc cggagtgctg agcgcagctc ccgcgatccc ctgtctgcgc gccgccgccg 420ccaagcccga gcccgagccg gggccgccgc caccggtgcc ggctccgagc ggcctcccgc 480gctccagccc gctgggagct gtccagtgct gaaaacccgc gcggacacag ccgatcgcgc 540ccggccggcc gcctccccgc accgagcccc gcgccggccg cgccatgccg cgctccttcc 600tggtaaagaa gatcaaaggg gacggcttcc agtgcagcgg ggtgccggcc cccacctacc 660accccttgga gacagcctac gtgctgcctg gcgcccgggg gcctcccggg gacaacgggt 720acgccccgca ccgcctgccc ccgagcagct acgatgcgga ccagaagccg ggcctggagc 780tggccccggc cgagcccgcg tacccgccgg cggcgccgga ggagtacagc gaccccgaaa 840gcccgcagtc gagcctgtcg gcgcgctact tccgagggga ggcggcagtg accgacagct 900actccatgga cgccttcttc atctcggacg ggcgctcgcg gcggcggcgg ggcgggggcg 960gcggggacgc ggggggctcg ggagacgcgg ggggcgccgg ggggcgcgcg gggcgcgcgg 1020gggcgcaggc gggcggcggg caccggcacg cgtgcgccga gtgcggcaag acctacgcca 1080cgtcgtcgaa cctgagccgc cacaagcaga cgcaccgcag cctggacagc cagctggcgc 1140gcaaatgccc gacgtgcggc aaggcctacg tgtccatgcc cgcgctcgcc atgcacctgc 1200tcacgcacaa cctgcgccac aagtgcggcg tctgcggcaa ggccttctcg cggccctggc 1260tgctgcaggg tcacatgcgc tcgcacaccg gcgaaaagcc gttcggctgc gcgcactgcg 1320gcaaggcctt cgccgaccgc tccaacctgc gcgcgcacat gcagacgcac tcggccttca 1380agcactaccg ctgccgccag tgcgacaaga gcttcgcgct caagtcctac ctccacaagc 1440actgcgaggc ggcctgcgcc aaggcggccg agccaccccc gccgaccccc gccggcccgg 1500ccagctgagc cttccgcctc gccctcgcgc ccggaactcg ctctccacgc gccccgggcc 1560ccctacctgc gcccgcagcg ccctcgccca gccccggctg cgtttccctg cccatgaccc 1620tctcgtgggg acccccggcc cggcccggaa cttttctccc caacccccaa acccacgact 1680cacttccaca ccgtcttccc cagcgtcccc cgacccactt catcctttcc ggccatccct 1740cggaccttga cccccctgtc cgcgccttca aggctcccaa ctcaggcacc aggtccgcac 1800ctctcccgcg ccccagagcc ggaaatttct ccctcccaga ccccgcgctg ctcttctaac 1860ttctagatct tttctctcgc ttctcagctg aatctctctg accattcctc tactggagcc 1920cccaatgaaa gccgcaaacc tatacccttg acgcccatac ctgcctcagt ttccccatcc 1980acacctggac gcaggccctg ccgccctgag accacagtcc aaccccagac catccctctg 2040gcctctgctg aatctcccag agtgtgtcca tctggggatc taatcttcac ccttcttgaa 2100gtctccgatc tgtccctgat cccatccatg ctcaggcctt aggtctggcc attcacacac 2160acacacaccc ccacacccca ctcccacaca cagttcctac tgtcttcagt ccatggggat 2220ctcatcaccc actcccagcc ccagctccag ctgccctcac ctctctagca gctctcaccc 2280ctctgagtct acctaacctg gagggggctt gaggggcact tcagggccct gccccctaat 2340gtaacccctc tctcaccccg gcacaacctg ggcttccaca gtctggagct gccctacccc 2400cagattctct gagcactttc ccccatcccc agggccacag atccccttcc ttacttgttg 2460gggggaggtc tggagcacct cgccaatgcc cttcccccat ccggctgggt ccttccagtt 2520atttatttgt gtatttattt atttatctat ttattattct atttaatctc ttggcctcac 2580ccagggaccg tctggcttcc ccagctggac tgggaggtca ggaagccagg caaggagagg 2640gacagcacag gccacagagg gctgccccca ccacacacac ccccgcgtct cgggagaaac 2700ccaccctctt cagagcctgc actcgctaag gaaggggact cgagaatggg ggggcagctt 2760gctgaccctc aactgggggg gtgagggagc atcaggaggt ccaggcgtgg ggcaggtccc 2820caccttcctg aaccttcccc tgccactcca ggcggcccgg agagaggagc ctcttcgatt 2880tgtttcctgt ttattcttct tcccgaggga ccccaggttc cagggaccga ctgagccccg 2940gacccgctgg ggcctctggc ctgctccccg ggaggggcgc tctctcccgg ccatgtctat 3000gcaactctcc cggacagagg ggccgggctg ccagccaccc gcccgttggc cgccactacc 3060tcctccgctg cgcttttgca tgcccgggcg aggaccgaag cacacacctc cacgcgcacg 3120ggccctggcc ccctccgctt taagcacacg cctctgcccc attgctctgc agccagcttg 3180ggggcaggag tcctggactc tcagatcctc ctctcccctc ctcttagcca caaaagaggg 3240gggagactgg aggcacccct gcagcagggg cacttcccta gggctccaga ggggtgaatg 3300ggcctttgct cagctccagg cccagcggga agggaggctg cagggatggt gggagggaga 3360ggggtagcag aagcaattat ggaggcgttg accctgtaaa tagcaacttc tgacagcaat 3420aattttccat gcatgctaag ccttttggcc atattttgta tgagcgcttg gcgctcctcc 3480tccgtcctat cccaactcac ccccaaaccc ccatcccttc cccacctcca gcagtattac 3540ttgtaacgca attcagggat attaaaggga ctttggccac tcaaaaaaaa aaaaaaaaaa 3600＜210〉5＜211〉3600＜212〉DNA＜213〉 (Homo sapiens)＜220〉＜221〉CDS＜222〉 (585).. (1505)＜400〉5gctttcttct ctctctgtta tctctcttgt cgtctttttg tccttatctt ggtctctctg 60cttctatttc tacctctctc agcgtgtgca gtctctcttt ccctgtatgt ctactgtctt 120tctccgagtc tctcttcttc tccccgcccc cctcactccc ctttctccgt ccctctctgt 180ctctgcgcct ttctcgccct cactttctcc cctcctgcct gctccgggcc tgcccccacc 240gcgcgccggt tcaggggaac gtgtgcggag cgattgtcct gggggacatt tgatggatta 300gagcgccgag cggttccatt gctaggggac cacctgatct ccagcctgcg tcccatataa 360agccggcagc cggagtgctg agcgcagctc ccgcgatccc ctgtctgcgc gccgccgccg 420ccaagcccga gcccgagccg gggccgccgc caccggtgcc ggctccgagc ggcctcccgc 480gctccagccc gctgggagct gtccagtgct gaaaacccgc gcggacacag ccgatcgcgc 540ccggccggcc gcctccccgc accgagcccc gcgccggccg cgcc atg ccg cgc tcc 596
Met?Pro?Arg?Ser
1ttc?ctg?gta?aag?aag?atc?aaa?ggg?gac?ggc?ttc?cag?tgc?agc?ggg?gtg??????644Phe?Leu?Val?Lys?Lys?Ile?Lys?Gly?Asp?Gly?Phe?Gln?Cys?Ser?Gly?Val5???????????????????10??????????????????15??????????????????20ccg?gcc?ccc?acc?tac?cac?ccc?ttg?gag?aca?gcc?tac?gtg?ctg?cct?ggc??????692Pro?Ala?Pro?Thr?Tyr?His?Pro?Leu?Glu?Thr?Ala?Tyr?Val?Leu?Pro?Gly
25??????????????????30??????????????????35gcc?cgg?ggg?cct?ccc?ggg?gac?aac?ggg?tac?gcc?ccg?cac?cgc?ctg?ccc??????740Ala?Arg?Gly?Pro?Pro?Gly?Asp?Asn?Gly?Tyr?Ala?Pro?His?Arg?Leu?Pro
40???????????????????45?????????????????50ccg?agc?agc?tac?gat?gcg?gac?cag?aag?ccg?ggc?ctg?gag?ctg?gcc?ccg?????788Pro?Ser?Ser?Tyr?Asp?Ala?Asp?Gln?Lys?Pro?Gly?Leu?Glu?Leu?Ala?Pro
55??????????????????60??????????????????65gcc?gag?ccc?gcg?tac?ccg?ccg?gcg?gcg?ccg?gag?gag?tac?agc?gac?ccc?????836Ala?Glu?Pro?Ala?Tyr?Pro?Pro?Ala?Ala?Pro?Glu?Glu?Tyr?Ser?Asp?Pro
70?????????????????75??????????????????80gaa?agc?ccg?cag?tcg?agc?ctg?tcg?gcg?cgc?tac?ttc?cga?ggg?gag?gcg?????884Glu?Ser?Pro?Gln?Ser?Ser?Leu?Ser?Ala?Arg?Tyr?Phe?Arg?Gly?Glu?Ala85??????????????????90??????????????????95??????????????????100gca?gtg?acc?gac?agc?tac?tcc?atg?gac?gcc?ttc?ttc?atc?tcg?gac?ggg?????932Ala?Val?Thr?Asp?Ser?Tyr?Ser?Met?Asp?Ala?Phe?Phe?Ile?Ser?Asp?Gly
105?????????????????110?????????????????115cgc?tcg?cgg?cgg?cgg?cgg?ggc?ggg?ggc?ggc?ggg?gac?gcg?ggg?ggc?tcg?????980Arg?Ser?Arg?Arg?Arg?Arg?Gly?Gly?Gly?Gly?Gly?Asp?Ala?Gly?Gly?Ser
120?????????????????125?????????????????130gga?gac?gcg?ggg?ggc?gcc?ggg?ggg?cgc?gcg?ggg?cgc?gcg?ggg?gcg?cag?????1028Gly?Asp?Ala?Gly?Gly?Ala?Gly?Gly?Arg?Ala?Gly?Arg?Ala?Gly?Ala?Gln
135?????????????????140?????????????????145gcg?ggc?ggc?ggg?cac?cgg?cac?gcg?tgc?gcc?gag?tgc?ggc?aag?acc?tac?????1076Ala?Gly?Gly?Gly?His?Arg?His?Ala?Cys?Ala?Glu?Cys?Gly?Lys?Thr?Tyr
150?????????????????155?????????????????160?gcc?acg?tcg?tcg?aac?ctg?agc?cgc?cac?aag?cag?acg?cac?cgc?agc?ctg?????1124Ala?Thr?Ser?Ser?Asn?Leu?Ser?Arg?His?Lys?Gln?Thr?His?Arg?Ser?Leu165?????????????????170?????????????????175?????????????????180gac?agc?cag?ctg?gcg?cgc?aaa?tgc?ccg?acg?tgc?ggc?aag?gcc?tac?gtg?????1172Asp?Ser?Gln?Leu?Ala?Arg?Lys?Cys?Pro?Thr?Cys?Gly?Lys?Ala?Tyr?Val
185?????????????????190?????????????????195tcc?atg?ccc?gcg?ctc?gcc?atg?cac?ctg?ctc?acg?cac?aac?ctg?cgc?cac?????1220Ser?Met?Pro?Ala?Leu?Ala?Met?His?Leu?Leu?Thr?His?Asn?Leu?Arg?His
200?????????????????205?????????????????210aag?tgc?ggc?gtc?tgc?ggc?aag?gcc?ttc?tcg?cgg?ccc?tgg?ctg?ctg?cag?????1268Lys?Cys?Gly?Val?Cys?Gly?Lys?Ala?Phe?Ser?Arg?Pro?Trp?Leu?Leu?Gln
215?????????????????220?????????????????225ggt?cac?atg?cgc?tcg?cac?acc?ggc?gaa?aag?ccg?ttc?ggc?tgc?gcg?cac?????1316Gly?His?Met?Arg?Ser?His?Thr?Gly?Glu?Lys?Pro?Phe?Gly?Cys?Ala?His
230?????????????????235?????????????????240tgc?ggc?aag?gcc?ttc?gcc?gac?cgc?tcc?aac?ctg?cgc?gcg?cac?atg?cag?????1364Cys?Gly?Lys?Ala?Phe?Ala?Asp?Arg?Ser?Asn?Leu?Arg?Ala?His?Met?Gln245?????????????????250?????????????????255?????????????????260acg?cac?tcg?gcc?ttc?aag?cac?tac?cgc?tgc?cgc?cag?tgc?gac?aag?agc?????1412Thr?His?Ser?Ala?Phe?Lys?His?Tyr?Arg?Cys?Arg?Gln?Cys?Asp?Lys?Ser
265?????????????????270?????????????????275ttc?gcg?ctc?aag?tcc?tac?ctc?cac?aag?cac?tgc?gag?gcg?gcc?tgc?gcc?????1460Phe?Ala?Leu?Lys?Ser?Tyr?Leu?His?Lys?His?Cys?Glu?Ala?Ala?Cys?Ala
280?????????????????285?????????????????290aag?gcg?gcc?gag?cca?ccc?ccg?ccg?acc?ccc?gcc?ggc?ccg?gcc?agc?????????1505Lys?Ala?Ala?Glu?Pro?Pro?Pro?Pro?Thr?Pro?Ala?Gly?Pro?Ala?Ser
295 300 305tgagccttcc gcctcgccct cgcgcccgga actcgctctc cacgcgcccc gggcccccta 1565cctgcgcccg cagcgccctc gcccagcccc ggctgcgttt ccctgcccat gaccctctcg 1625tggggacccc cggcccggcc cggaactttt ctccccaacc cccaaaccca cgactcactt 1685ccacaccgtc ttccccagcg tcccccgacc cacttcatcc tttccggcca tccctcggac 1745cttgaccccc ctgtccgcgc cttcaaggct cccaactcag gcaccaggtc cgcacctctc 1805ccgcgcccca gagccggaaa tttctccctc ccagaccccg cgctgctctt ctaacttcta 1865gatcttttct ctcgcttctc agctgaatct ctctgaccat tcctctactg gagcccccaa 1925tgaaagccgc aaacctatac ccttgacgcc catacctgcc tcagtttccc catccacacc 1985tggacgcagg ccctgccgcc ctgagaccac agtccaaccc cagaccatcc ctctggcctc 2045tgctgaatct cccagagtgt gtccatctgg ggatctaatc ttcacccttc ttgaagtctc 2105cgatctgtcc ctgatcccat ccatgctcag gccttaggtc tggccattca cacacacaca 2165cacccccaca ccccactccc acacacagtt cctactgtct tcagtccatg gggatctcat 2225cacccactcc cagccccagc tccagctgcc ctcacctctc tagcagctct cacccctctg 2285agtctaccta acctggaggg ggcttgaggg gcacttcagg gccctgcccc ctaatgtaac 2345ccctctctca ccccggcaca acctgggctt ccacagtctg gagctgccct acccccagat 2405tctctgagca ctttccccca tccccagggc cacagatccc cttccttact tgttgggggg 2465aggtctggag cacctcgcca atgcccttcc cccatccggc tgggtccttc cagttattta 2525tttgtgtatt tatttattta tctatttatt attctattta atctcttggc ctcacccagg 2585gaccgtctgg cttccccagc tggactggga ggtcaggaag ccaggcaagg agagggacag 2645cacaggccac agagggctgc ccccaccaca cacacccccg cgtctcggga gaaacccacc 2705ctcttcagag cctgcactcg ctaaggaagg ggactcgaga atgggggggc agcttgctga 2765ccctcaactg ggggggtgag ggagcatcag gaggtccagg cgtggggcag gtccccacct 2825tcctgaacct tcccctgcca ctccaggcgg cccggagaga ggagcctctt cgatttgttt 2885cctgtttatt cttcttcccg agggacccca ggttccaggg accgactgag ccccggaccc 2945gctggggcct ctggcctgct ccccgggagg ggcgctctct cccggccatg tctatgcaac 3005tctcccggac agaggggccg ggctgccagc cacccgcccg ttggccgcca ctacctcctc 3065cgctgcgctt ttgcatgccc gggcgaggac cgaagcacac acctccacgc gcacgggccc 3125tggccccctc cgctttaagc acacgcctct gccccattgc tctgcagcca gcttgggggc 3185aggagtcctg gactctcaga tcctcctctc ccctcctctt agccacaaaa gaggggggag 3245actggaggca cccctgcagc aggggcactt ccctagggct ccagaggggt gaatgggcct 3305ttgctcagct ccaggcccag cgggaaggga ggctgcaggg atggtgggag ggagaggggt 3365agcagaagca attatggagg cgttgaccct gtaaatagca acttctgaca gcaataattt 3425tccatgcatg ctaagccttt tggccatatt ttgtatgagc gcttggcgct cctcctccgt 3485cctatcccaa ctcaccccca aacccccatc ccttccccac ctccagcagt attacttgta 3545acgcaattca gggatattaa agggactttg gccactcaaa aaaaaaaaaa aaaaa 3600＜210〉6＜211〉307＜212〉PRT＜213〉 ( Homo sapiens )＜400〉6Met Pro Arg Ser Phe Leu Val Lys Lys Ile Lys Gly Asp Gly Phe Gln1 5 10 15Cys Ser Gly Val Pro Ala Pro Thr Tyr His Pro Leu Glu Thr Ala Tyr
20?????????????????25??????????????????30Val?Leu?Pro?Gly?Ala?Arg?Gly?Pro?Pro?Gly?Asp?Asn?Gly?Tyr?Ala?Pro
35??????????????????40??????????????????45His?Arg?Leu?Pro?Pro?Ser?Ser?Tyr?Asp?Ala?Asp?Gln?Lys?Pro?Gly?Leu
50??????????????????55??????????????????60Glu?Leu?Ala?Pro?Ala?Glu?Pro?Ala?Tyr?Pro?Pro?Ala?Ala?Pro?Glu?Glu65??????????????????70??????????????????75???????????????????80Tyr?Ser?Asp?Pro?Glu?Ser?Pro?Gln?Ser?Ser?Leu?Ser?Ala?Arg?Tyr?Phe
85??????????????????90??????????????????95Arg?Gly?Glu?Ala?Ala?Val?Thr?Asp?Ser?Tyr?Ser?Met?Asp?Ala?Phe?Phe
100?????????????????105?????????????????110Ile?Ser?Asp?Gly?Arg?Ser?Arg?Arg?Arg?Arg?Gly?Gly?Gly?Gly?Gly?Asp
115?????????????????120?????????????????125Ala?Gly?Gly?Ser?Gly?Asp?Ala?Gly?Gly?Ala?Gly?Gly?Arg?Ala?Gly?Arg
130?????????????????135?????????????????140Ala?Gly?Ala?Gln?Ala?Gly?Gly?Gly?His?Arg?His?Ala?Cys?Ala?Glu?Cys145?????????????????150?????????????????155?????????????????160Gly?Lys?Thr?Tyr?Ala?Thr?Ser?Ser?Asn?Leu?Ser?Arg?His?Lys?Gln?Thr
165?????????????????170?????????????????175His?Arg?Ser?Leu?Asp?Ser?Gln?Leu?Ala?Arg?Lys?Cys?Pro?Thr?Cys?Gly
180?????????????????185?????????????????190Lys?Ala?Tyr?Val?Ser?Met?Pro?Ala?Leu?Ala?Met?His?Leu?Leu?Thr?His
195?????????????????200?????????????????205Ash?Leu?Arg?His?Lys?Cys?Gly?Val?Cys?Gly?Lys?Ala?Phe?Ser?Arg?Pro
210?????????????????215?????????????????220Trp?Leu?Leu?Gln?Gly?His?Met?Arg?Ser?His?Thr?Gly?Glu?Lys?Pro?Phe225?????????????????230?????????????????235?????????????????240Gly?Cys?Ala?His?Cys?Gly?Lys?Ala?Phe?Ala?Asp?Arg?Ser?Asn?Leu?Arg
245?????????????????250?????????????????255Ala?His?Met?Gln?Thr?His?Ser?Ala?Phe?Lys?His?Tyr?Arg?Cys?Arg?Gln
260?????????????????265?????????????????270Cys?Asp?Lys?Ser?Phe?Ala?Leu?Lys?Ser?Tyr?Leu?His?Lys?His?Cys?Glu
275?????????????????280?????????????????285Ala?Ala?Cys?Ala?Lys?Ala?Ala?Glu?Pro?Pro?Pro?Pro?Thr?Pro?Ala?Gly
290 295 300Pro Ala Ser305＜210〉7＜211〉3491＜212〉DNA＜213〉 ( Homo sapiens )＜400〉7gacccccatc acccccaggt cgccctgcag gcactgtgtg tttggacagc tggaagcaga 60gctcggcctg tgacctgcct ggtccagcaa ctggaccttc tggaggtgag cctgggagtg 120gccgagcaga ggaaatcacc cccatgggag gtaggggagg ggtgaggccc cccccaccag 180gttctggaat gttccgtggg ctggatcctg gagaacccac ttctctgggc tccagaaagg 240caagggcaat gtttctttga cctgagaaag caaagcagcc ccccgacatc cttccacaca 300ctcatcccct ctggtgggga gccgggggga cacatatgag atggacaggg tgggggccca 360caccccaggc tactcggaca gcaggggtca atcccgacgg ctggagaaac ctcaggcccc 420aagtgcacca atggcccaga tggctggaga cctcttcctt tgtctcctct gtctccaaga 480tccaggagcc caggctcagc ctgcgaacca cctggccctg gccctggagg gggatgggga 540gggtctgtga ccatcctggg gggtatctgc gcccccttct tctacctggg gacaagggcg 600cgaagtccca tgcagccccc atgcagcctc acgtgcctga cgatgggagc caggccagcc 660cctcacacgg gtgctcctgg gcagcctctc tggtagcctg gacgagtcac cccaggggca 720catgctccag gcagccccct cacctcaggg ggcatgggag gcgctggggg tcttcagctg 780gtgatggggt gactgacgag gatcagcccc aaccccgggg tcctctggcc gctggcaacc 840ggagcactcc cccagttgga gctgaaccat gactccagtc cttcccaaga gatggtgggg 900aatcgtcacc atggctggag tcccgcagac tgggcaggcc agacaggccc ttggaggagg 960ctggtgggat ccgagtgggc agaggggaga cggaggaggg aaaggcagcc ccagcctgcg 1020gtggtgcttg tcctgggcgg gaggggccct gctgtcactg acgtgggaac caccgagtca 1080ccggaggggc aggggcagag gggtctcggt ggaaaggggg tccccagagg ccttgggaga 1140cgggggaggt gcctagggct ctctggcctt ggtctctacc agctcaacga gggctttgtt 1200ccaggggccc ctgggtccct ctgcctccaa caatctgggc tgcgttttct gagcacctac 1260tgtgtgctgg gtgtgggcgt ccgggcaggc actgactaag ccttgtccct ccccagagtc 1320acagtgagac acacagccac atcctgtaaa tgaagacctc gtggctcaga gaggtcaggt 1380aacttcccca aagcctgtct ggcctcccag caccatccct agtcccagag ggaggtggaa 1440tgaaggcagg gctaaggcca ggctgcatgg aggcagaggc acctgccggc ctgcaggacc 1500aaaggttcta ttggaggcag tgcctgggca gacaaaggcc ctcagattca caagctgaag 1560ccctagcacc cagggcccca gtgtgctact gtgtttggag acaggtaaag gttgtcaagg 1620taagacaggg tggccctgat ccaatatgac tggcgtcctt gtaagaagag gagattggga 1680cagacacgcc ccgagggatg gccatgtgag ggcgcctcag gaagacggtg tgcaagagcg 1740ggaagcgggg cctcggaaga gccagccctt ggatctcggg cttccaggag aatcagttcc 1800tgcagttgaa gccaccaggg tctgtgcaac tttgtcacag ctgcccgagc tgacaaattc 1860acctgactta agccccccgc cccacccgac cacagtgtca cccagatgct tggcaggtgc 1920tgtggcaggg gctgggtccc tgctcgacct tcgggtgccg gggtgtcccg tataagagca 1980gcacagccac cgccacggcc actcgcagtt ccaggctccg ggcagcatct gggaccccag 2040ttcctgttgt gagaggctcc ccactgccat cccaccccct gcaaggtgcc tcggagagcg 2100gctggaggct gacgggtcga ggcctgggtt ttacggtcct gcctgggttt ctgtgtggcc 2160tggggctgcc taccatcttt ccctctctgg tcccccctac tctcatatac caggctgggt 2220gactggggcc caggagcggc ccccccctcc tctgggcttc ctggtggcac ccctggcctg 2280acagcccctg ggaaggcctc ccctctccat ctcggggccc gagcccggcc ccacctgctg 2340agctgcagcc tccaaggcct ccttcgcacg cgtgtcagct ctgacctcag ggctccccac 2400gcgtgttccc ctctaggaag gcccccccag gggctcccca aaatcctgtc ctccctgaag 2460cacctgttct tcgtgctcct ccatgagggc ccgcacgggg ccgctgaccg cagtctcagt 2520cacagcctgg gagcgcctgg aggttggggg gcagcaggcg ctcagtcagt cctcgggtca 2580ccatggatgg ccttgctgtg tgatccacac tgaggcccag tggtcagctg gggggctgga 2640caggaggcag ccacttcctg gcctggctgg tgactcacag gcccgtttct gggaaaactc 2700ccacccaccc ccactcaatc cggatggcgc agagctcgct gtggggacca gctgtgggaa 2760gtgcattgat gattaggggc cgggtctggg ggtggggtaa ggcgggagcc ggggcggagg 2820gtggggtggg gttgttggga cgtctccacc agaygcctgc ctgggctgct gagctgtgca 2880tcccgtgtgc cctgacgctc cacatgctcc ctcaccgggc aggggtcggs ggccccgaat 2940atcatcacag sgagaccaca cgaygcttcc tgcctgaacc ccttcctcag gtccctcacg 3000cttcaagggg tgcctggccc cagcctcgcc ttgcccagcc ttgccaaggc cagcacagcc 3060caggagtaag ccagagaaaa gaaaatgcca ttcggaaaat cctaaggaag agaggatgca 3120tttcctcgtc tccacgtgga agcggatcac cctaaaggtc ttcatcctgg tctttttcat 3180gctgaggagg ctggggagga gaaggaagag gaggggctgg tcttgctgac ccaggagcag 3240cagagaaagg agaaaatcca cgtgcagggg ctggccagcg ctgggcttct cgggaatgga 3300atcacacacg gactgtggcg tacacatcac aactttaaac cccgtccgta ctgaagcctg 3360cgtccgaatt tcttctgttt ttgtggcgga aggataaata ttccattgta aaaaaaaaaa 3420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3480aaaaaaaaaa a 3491＜210〉8＜211〉3491＜212〉DNA＜213〉 ( Homo sapiens )＜220〉＜221〉CDS＜222〉 ( 336 ) .. ( 869 )＜400〉8gacccccatc acccccaggt cgccctgcag gcactgtgtg tttggacagc tggaagcaga 60gctcggcctg tgacctgcct ggtccagcaa ctggaccttc tggaggtgag cctgggagtg 120gccgagcaga ggaaatcacc cccatgggag gtaggggagg ggtgaggccc cccccaccag 180gttctggaat gttccgtggg ctggatcctg gagaacccac ttctctgggc tccagaaagg 240caagggcaat gtttctttga cctgagaaag caaagcagcc ccccgacatc cttccacaca 300ctcatcccct ctggtgggga gccgggggga cacat atg aga tgg aca ggg tgg 353
Met?Arg?Trp?Thr?Gly?Trp
1???????????????5ggg?ccc?aca?ccc?cag?gct?act?cgg?aca?gca?ggg?gtc?aat?ccc?gac?ggc??????401Gly?Pro?Thr?Pro?Gln?Ala?Thr?Arg?Thr?Ala?Gly?Val?Asn?Pro?Asp?Gly
10??????????????????15??????????????????20tgg?aga?aac?ctc?agg?ccc?caa?gtg?cac?caa?tgg?ccc?aga?tgg?ctg?gag??????449Trp?Arg?Asn?Leu?Arg?Pro?Gln?Val?His?Gln?Trp?Pro?Arg?Trp?Leu?Glu
25??????????????????30??????????????????35acc?tct?tcc?ttt?gtc?tcc?tct?gtc?tcc?aag?atc?cag?gag?ccc?agg?ctc??????497Thr?Ser?Ser?Phe?Val?Ser?Ser?Val?Ser?Lys?Ile?Gln?Glu?Pro?Arg?Leu
40??????????????????45??????????????????50agc?ctg?cga?acc?acc?tgg?ccc?tgg?ccc?tgg?agg?ggg?atg?ggg?agg?gtc??????545Ser?Leu?Arg?Thr?Thr?Trp?Pro?Trp?Pro?Trp?Arg?Gly?Met?Gly?Arg?Val55??????????????????60??????????????????65??????????????????70tgt?gac?cat?cct?ggg?ggg?tat?ctg?cgc?ccc?ctt?ctt?cta?cct?ggg?gac??????593Cys?Asp?His?Pro?Gly?Gly?Tyr?Leu?Arg?Pro?Leu?Leu?Leu?Pro?Gly?Asp
75??????????????????80??????????????????85aag?ggc?gcg?aag?tcc?cat?gca?gcc?ccc?atg?cag?cct?cac?gtg?cct?gac??????641Lys?Gly?Ala?Lys?Ser?His?Ala?Ala?Pro?Met?Gln?Pro?His?Val?Pro?Asp
90??????????????????95??????????????????100gat?ggg?agc?cag?gcc?agc?ccc?tca?cac?ggg?tgc?tcc?tgg?gca?gcc?tct??????689Asp?Gly?Ser?Gln?Ala?Ser?Pro?Ser?His?Gly?Cys?Ser?Trp?Ala?Ala?Ser
105?????????????????110?????????????????115ctg?gta?gcc?tgg?acg?agt?cac?ccc?agg?ggc?aca?tgc?tcc?agg?cag?ccc??????737Leu?Val?Ala?Trp?Thr?Ser?His?Pro?Arg?Gly?Thr?Cys?Ser?Arg?Gln?Pro
120?????????????????125?????????????????130cct?cac?ctc?agg?ggg?cat?ggg?agg?cgc?tgg?ggg?tct?tca?gct?ggt?gat??????785Pro?His?Leu?Arg?Gly?His?Gly?Arg?Arg?Trp?Gly?Ser?Ser?Ala?Gly?Asp135?????????????????140?????????????????145?????????????????150ggg?gtg?act?gac?gag?gat?cag?ccc?caa?ccc?cgg?ggt?cct?ctg?gcc?gct??????833Gly?Val?Thr?Asp?Glu?Asp?Gln?Pro?Gln?Pro?Arg?Gly?Pro?Leu?Ala?Ala
155?????????????????160?????????????????165ggc?aac?cgg?agc?act?ccc?cca?gtt?gga?gct?gaa?cca?tgactccagt???????????879Gly?Asn?Arg?Ser?Thr?Pro?Pro?Val?Gly?Ala?Glu?Pro
170 175ccttcccaag agatggtggg gaatcgtcac catggctgga gtcccgcaga ctgggcaggc 939cagacaggcc cttggaggag gctggtggga tccgagtggg cagaggggag acggaggagg 999gaaaggcagc cccagcctgc ggtggtgctt gtcctgggcg ggaggggccc tgctgtcact 1059gacgtgggaa ccaccgagtc accggagggg caggggcaga ggggtctcgg tggaaagggg 1119gtccccagag gccttgggag acgggggagg tgcctagggc tctctggcct tggtctctac 1179cagctcaacg agggctttgt tccaggggcc cctgggtccc tctgcctcca acaatctggg 1239ctgcgttttc tgagcaccta ctgtgtgctg ggtgtgggcg tccgggcagg cactgactaa 1299gccttgtccc tccccagagt cacagtgaga cacacagcca catcctgtaa atgaagacct 1359cgtggctcag agaggtcagg taacttcccc aaagcctgtc tggcctccca gcaccatccc 1419tagtcccaga gggaggtgga atgaaggcag ggctaaggcc aggctgcatg gaggcagagg 1479cacctgccgg cctgcaggac caaaggttct attggaggca gtgcctgggc agacaaaggc 1539cctcagattc acaagctgaa gccctagcac ccagggcccc agtgtgctac tgtgtttgga 1599gacaggtaaa ggttgtcaag gtaagacagg gtggccctga tccaatatga ctggcgtcct 1659tgtaagaaga ggagattggg acagacacgc cccgagggat ggccatgtga gggcgcctca 1719ggaagacggt gtgcaagagc gggaagcggg gcctcggaag agccagccct tggatctcgg 1779gcttccagga gaatcagttc ctgcagttga agccaccagg gtctgtgcaa ctttgtcaca 1839gctgcccgag ctgacaaatt cacctgactt aagccccccg ccccacccga ccacagtgtc 1899acccagatgc ttggcaggtg ctgtggcagg ggctgggtcc ctgctcgacc ttcgggtgcc 1959ggggtgtccc gtataagagc agcacagcca ccgccacggc cactcgcagt tccaggctcc 2019gggcagcatc tgggacccca gttcctgttg tgagaggctc cccactgcca tcccaccccc 2079tgcaaggtgc ctcggagagc ggctggaggc tgacgggtcg aggcctgggt tttacggtcc 2139tgcctgggtt tctgtgtggc ctggggctgc ctaccatctt tccctctctg gtccccccta 2199ctctcatata ccaggctggg tgactggggc ccaggagcgg cccccccctc ctctgggctt 2259cctggtggca cccctggcct gacagcccct gggaaggcct cccctctcca tctcggggcc 2319cgagcccggc cccacctgct gagctgcagc ctccaaggcc tccttcgcac gcgtgtcagc 2379tctgacctca gggctcccca cgcgtgttcc cctctaggaa ggccccccca ggggctcccc 2439aaaatcctgt cctccctgaa gcacctgttc ttcgtgctcc tccatgaggg cccgcacggg 2499gccgctgacc gcagtctcag tcacagcctg ggagcgcctg gaggttgggg ggcagcaggc 2559gctcagtcag tcctcgggtc accatggatg gccttgctgt gtgatccaca ctgaggccca 2619gtggtcagct ggggggctgg acaggaggca gccacttcct ggcctggctg gtgactcaca 2679ggcccgtttc tgggaaaact cccacccacc cccactcaat ccggatggcg cagagctcgc 2739tgtggggacc agctgtggga agtgcattga tgattagggg ccgggtctgg gggtggggta 2799aggcgggagc cggggcggag ggtggggtgg ggttgttggg acgtctccac cagaygcctg 2859cctgggctgc tgagctgtgc atcccgtgtg ccctgacgct ccacatgctc cctcaccggg 2919caggggtcgg sggccccgaa tatcatcaca gsgagaccac acgaygcttc ctgcctgaac 2979cccttcctca ggtccctcac gcttcaaggg gtgcctggcc ccagcctcgc cttgcccagc 3039cttgccaagg ccagcacagc ccaggagtaa gccagagaaa agaaaatgcc attcggaaaa 3099tcctaaggaa gagaggatgc atttcctcgt ctccacgtgg aagcggatca ccctaaaggt 3159cttcatcctg gtctttttca tgctgaggag gctggggagg agaaggaaga ggaggggctg 3219gtcttgctga cccaggagca gcagagaaag gagaaaatcc acgtgcaggg gctggccagc 3279gctgggcttc tcgggaatgg aatcacacac ggactgtggc gtacacatca caactttaaa 3339ccccgtccgt actgaagcct gcgtccgaat ttcttctgtt tttgtggcgg aaggataaat 3399attccattgt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3459aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 3491＜210〉9＜211〉178＜212〉PRT＜213〉 ( Homo sapiens )＜400〉9Met Arg Trp Thr Gly Trp Gly Pro Thr Pro Gln Ala Thr Arg Thr Ala1 5 10 15Gly Val Asn Pro Asp Gly Trp Arg Asn Leu Arg Pro Gln Val His Gln
20??????????????????25??????????????????30Trp?Pro?Arg?Trp?Leu?Glu?Thr?Ser?Ser?Phe?Val?Ser?Ser?Val?Ser?Lys
35??????????????????40??????????????????45Ile?Gln?Glu?Pro?Arg?Leu?Ser?Leu?Arg?Thr?Thr?Trp?Pro?Trp?Pro?Trp
50??????????????????55??????????????????60Arg?Gly?Met?Gly?Arg?Val?Cys?Asp?His?Pro?Gly?Gly?Tyr?Leu?Arg?Pro65??????????????????70??????????????????75??????????????????80Leu?Leu?Leu?Pro?Gly?Asp?Lys?Gly?Ala?Lys?Ser?His?Ala?Ala?Pro?Met
85??????????????????90??????????????????95Gln?Pro?His?Val?Pro?Asp?Asp?Gly?Ser?Gln?Ala?Ser?Pro?Ser?His?Gly
100?????????????????105?????????????????110Cys?Ser?Trp?Ala?Ala?Ser?Leu?Val?Ala?Trp?Thr?Ser?His?Pro?Arg?Gly
115?????????????????120?????????????????125Thr?Cys?Ser?Arg?Gln?Pro?Pro?His?Leu?Arg?Gly?His?Gly?Arg?Arg?Trp
130?????????????????135?????????????????140Gly?Ser?Ser?Ala?Gly?Asp?Gly?Val?Thr?Asp?Glu?Asp?Gln?Pro?Gln?Pro145?????????????????150?????????????????155?????????????????160Arg?Gly?Pro?Leu?Ala?Ala?Gly?Asn?Arg?Ser?Thr?Pro?Pro?Val?Gly?Ala
165 170 175Glu Pro＜210〉10＜211〉20＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉10tgtgaacacc ttgagccttg 20＜210〉11＜211〉18＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉11tgcaacctct gcctcaca 18＜210〉12＜211〉20＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉12tcagcgtgtg cagtctctct 20＜210〉13＜211〉20＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉13tttttgagtg gccaaagtcc 20＜210〉14＜211〉20＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉14gcactgtgtg tttggacagc 20＜210〉15＜211〉20＜212〉DNA＜213〉＜220〉＜221〉misc feature＜223〉＜400〉15tatccttccg ccacaaaaac 20
Claims (10)
1. isolating people's albumen with promotion 3T3 cell transformation function is characterized in that it comprises the polypeptide of the aminoacid sequence with the group of being selected from down: SEQ ID NO:3,6,9;
Or its conservative property variation polypeptide or its active fragments or its reactive derivative.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with aminoacid sequence of the group of being selected from down: SEQ ID NO:3,6,9.
3. isolating polynucleotide is characterized in that, 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:
(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;
(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:3,6,9.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down group:
SEQ ID NO:2,5,8 coding region sequence or full length sequence.
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 3T3 cell transformation function is characterized in that this method comprises:
(a) being fit to express under the proteic condition of people with promotion 3T3 cell transformation function, cultivate the described host cell of claim 7;
(b) from culture, isolate polypeptide with the people's protein-active that promotes 3T3 cell transformation function.
9. an energy and claim 1 are described has the people's protein-specific bonded antibody that promotes 3T3 cell transformation function.
10. nucleic acid molecule, it contains a successive 15-1000 Nucleotide in the described polynucleotide of claim 3.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021116458A CN1190446C (en) | 2002-05-13 | 2002-05-13 | New human protein with function of improving mouse NIH/3T3 cell transformation and its encoding sequence |
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| CN1190446C CN1190446C (en) | 2005-02-23 |
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