CN1297931A - Human ABC transfer protein 39 as one new kind of polypeptide and polynucleotides encoding this polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, human ABC transfer protein 39, polynucleotides encoding this polypeptide and DNA recombination process to produce the polypeptide. The present invention also discloses the method of applying the polypeptide in treating various diseases, such as malignant tumor, nosohemia, HIV infection, immunological diseases and inflammations. The present invention also discloses the antagonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this polypeptide.

Description

A kind of new polypeptide-human abc transport protein 39 and the polynucleotide of this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide-human abc transport protein 39, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

ATP is conjugated protein extensively to be existed in vivo, and has very important physiologic function, and the propagation, signal transduction, the albumen that relate to cell synthesize critical functions such as reaching the albumen location.ATP is conjugated protein can be divided into various super families again, and each super family has numerous family members again.The member of ATP protein family is all contained conservative ATP combined function territory, and ATP combined function territory is used as molecular switch in vivo, whether combines with ADP, ATP by it and regulates proteic expression and do not express.Thereby, ATP is conjugated protein very important effect in vivo, its abnormal expression will cause histiocytic abnormality proliferation and abnormal exprssion, thereby cause various associated diseases, as: various malignant tumours and cancer, various growth disorders, various disease of immune system etc.

ATP combined function territory be various ATP conjugated protein in ubiquitous structural domain, some albumen that connect ATP all more or less contain this conservative mot if sequence, and this motif has conservative characteristic sequential fragments: [AG]-X (4)-G-K-[ST] (wherein X is an arbitrary amino acid residue).What guard the most among these motif is the glycine rich region, and this zone forms a flexible ring between β line and α spiral.This crooked ring usually and a certain phosphate group reaction on the Nucleotide, this motif sequence is commonly called " A " consensus sequence or P ring.This motif is responsible for combining with ATP in vivo usually, for albumen performance biological function provides required energy and stabilize proteins structure in vivo, assists albumen to finish its normal physiologic function.Its abnormal expression will directly cause some abnormal exprssion and dysfunction, thereby cause various relevant diseases, as grow generation of disorder, tumour and cancer etc.Thereby this motif plays a part very important in albumen is brought into normal play the process of physiological function.

In eucaryon and prokaryotic organism, there is a class ATP conjugated protein, be called as abc transport albumen (ATP-binding cassette transporter), different abc transport albumen has been formed an important protein family.The crystalline structure of the proteic ATP binding subunit of some abc transport is found, and concrete structure sees also pertinent literature.(Nature?1998?Dec?17；396(6712)：703-7)

There are some researches show that the proteic basic role of abc transport is transhipment and the release of regulation and control ATP, kinetic mechanism wherein is revealed, thus can clear and definite abc transport albumen plays an important role in cell need utilize the vital process of energy.(Methods?Enzymol.1998；292：664-75)(Am?J?Physiol?1999Jan；276(1?Pt?1)：C1-8)

According to research, the abc transport albumen that is in the organism of different steps in system's generating process is fully conservative on evolving, and it has vital role for the transhipment of some small molecules (as maltose, trehalose etc.).(J?Bacteriol?1998?Feb；180(3)：680-9)

If abc transport more proteic members undergo mutation, can cause the appearance of a lot of diseases, be proved to be as bladder cystic fibrosis, hyperinsulinemia, StargardtShi disease, combination drug opposing disease or the like this point, and the abc transport proteic encoding gene relevant with above-mentioned some diseases cloned.(HumMol?Genet1998?Feb；7：203-7)；(Biochem?Biophys?Res?Commun.1997?Oct?9；239(1)：261-4)

Find that after deliberation several abc transport albumen (ALDP, ALDR are arranged, PMP70) in the membrane structure of people's peroxysome, have and exist and play an important role, wherein, the sudden change of ALDP can cause the leukodystrophy of X-linkage, and the function of ALDR and PMP70 is not clear and definite as yet.(Hum?Mol?Genet.1997Oct；6(11)：1925-31)

The helABC genes encoding the proteic mixture of a kind of abc transport, help the biosynthesizing of cytochrome c that sufficient regulating and controlling effect is arranged for the transhipment of heme.(J?Mol?Biol?1997?May16；268(4)：724-38)

AbcA is a kind of abc transport albumen, and result of study shows that it can influence the transport process of more needed molecules in the cell chemotaxis mechanism.(J?Bacteriol?1998?Nov；180(21)：5697-703)

The Histidine permease also is a kind of abc transport albumen, and it is incorporated on the film, constitutes a mixture, can hydrolysising ATP when the part soluble receptors exists, have the activity of ATPase enzyme.(Proc?Natl?AcadSci?USA?1998?Mar?31；95(7)：3495-500)

In addition, comprised also in the abc transport albumen that a lot of and medical science has the albumen of important relation, for example, the bladder cystic fibrosis is striden film regulatory factor (CFTR), P-glycoprotein or the like.

According to amino acid homology result relatively, polypeptide of the present invention is accredited as HABCT39 (HABCT39) by deduction, and its homologous protein is the abc transport albumen of fission yeast, and its albumen number is AL022299.

Because HABCT39's albumen plays an important role in regulating body critical functions such as cell fission and fetal development as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify HABCT39's albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new HABCT39's protein coding gene also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

An object of the present invention is to provide isolating new polypeptide-human abc transport protein 39 with and fragment, analogue and derivative.

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

Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the HABCT39 that encodes.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the HABCT39 that encodes.

Another object of the present invention provides the method for producing the HABCT39.

Another object of the present invention provides the antibody at polypeptide-human abc transport protein 39 of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor at polypeptide-human abc transport protein 39 of the present invention.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with the HABCT39.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;

(b) with polynucleotide (a) complementary polynucleotide;

(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 70% homogeny.

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 685-1746 position among the SEQ ID NO:1; (b) has the sequence of 1-2049 position among the SEQ ID NO:1.

The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for the compound of HABCT39's protein-active, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with HABCT39's abnormal protein, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of cancer, developmental character disease or immunological disease or other purposes owing to the medicine of HABCT39's disease that abnormal expression causes in preparation.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

The following term of using in this specification sheets and claims has following implication unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when combining with the HABCT39, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with HABCT39's molecule.

" antagonist " or " inhibition " be meant when combining with the HABCT39, a kind ofly seals or regulate HABCT39's the biologic activity or the molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with HABCT39's molecule.

" adjusting " is meant that HABCT39's function changes, and comprises the change of the change of the rising of protein active or reduction, binding characteristic and HABCT39's any other biological property, function or immune property.

" pure basically " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying HABCT39 of standard.Basically pure HABCT39 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of HABCT39's polypeptide is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergenesoftware package, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula:

The residue number of mating between sequence A and the sequence B

100

Interval residue number in the residue number-sequence B of interval in the residue number-sequence A of sequence A

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fa, F (ab ') ₂And Fv, its energy specificity is in conjunction with HABCT39's antigenic determinant.

" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

Many As used hereins, " isolating " are 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 peptide, 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 HABCT39 " is meant that the HABCT39 is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying HABCT39 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of HABCT39's polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide-human abc transport protein 39, it is made up of the aminoacid sequence shown in the SEQ IDNO:2 basically.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 HABCT39's fragment, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of HABCT39 of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 2049 bases, its open reading frame (685-1746) 353 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the abc transport albumen of this polypeptide and fission yeast has 53% homology, deducibility goes out the 26S Proteasome Structure and Function that this HABCT39 has the abc transport protein similar of fission yeast.

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.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only 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 " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, 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 the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.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 adds and to use 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 sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate coding HABCT39's polynucleotide.

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

Coding HABCT39's of the present invention special polynucleotide sequence can obtain with several different methods.For example, separate polynucleotide with hybridization technique 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 homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

Sequence dna fragment of the present invention also 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 described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of 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., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of mensuration HABCT39's transcript; (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 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of HABCT39's genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

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 from the library, obtain the cDNA of total length, 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 polynucleotide 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 polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.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 directly with the host cell of HABCT39's encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, coding HABCT39's polynucleotide sequence can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor 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 may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna that contains the HABCT39 that encodes and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory 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; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, 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.

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 etc.

Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.

Among the present invention, coding HABCT39's polynucleotide or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.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, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce HABCT39 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of everybody abc transport protein 39 of coding of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). in suitable medium, cultivate host cell;

(3). separation, protein purification from substratum or cell.

In step (2), 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.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.

It is conjugated protein that polypeptide of the present invention (HABCT39) belongs to ATP, and its effect comprises the transhipment that promotes ATP, can play an important role in cell need utilize the vital process of energy; Polypeptide of the present invention has the activity of ATPase enzyme, and certain ATP hydrolysis function is arranged; It has vital role for some micromolecular transhipment in the organism; It is inferred existence and is played an important role in the membrane structure of people's peroxysome; It has sufficient regulating and controlling effect for the biosynthesizing of cytochrome c; Undergo mutation as polypeptide of the present invention, may cause the appearance of a lot of diseases, resist leukodystrophy of disease, X-linkage or the like as bladder cystic fibrosis, hyperinsulinemia, StargardtShi disease, combination drug.

Polypeptide of the present invention can be used for the diagnosis and the treatment of a lot of diseases, except that above-mentioned several diseases, also comprises: malignant tumour, endocrine system disease, nervous system disorders, immunological disease, people's acquired immune deficiency syndrome (AIDS) or the like.

Utilize the treatable growth disorders of polypeptide of the present invention to comprise: spina bifida, the cranium fissure, anencephalia, the brain bulging, the hole deformity of brain, the Down syndromes, congenital hydrocephalus, the aqueduct deformity, achondroplastic dwarf's disease, spondyloepiphyseal dysplasia disease, pseudocartilage underdevelopment disease, the Langer-Giedion syndromes, chonechondrosteron, hypogenitalism, adrenal,congenital hyperplasia, epispadia, latent, with malformation syndrome of short and small stature (as Conradi syndromes and Danbolt-Closs syndromes), congenital glaucoma or cataract, congenital lens position is unusual, congenital little fissura palpebrae, retinal development is unusual, atrophia nervi optici congenita, congenital sensory nerve hearing loss, ulcuscuris, monster, the Williams syndromes, the Alagille syndromes, shellfish syndrome Weis two or the like.

Utilize the treatable various tumours of polypeptide of the present invention to comprise: to comprise that epithelium is (as the substrate epithelium, squama shape epithelium, myxocyte or the like), (as fibrous tissue, fatty tissue, cartilaginous tissue, smooth muscle tissue, blood vessel and lymphatic endothelial tissue or the like), hemopoietic tissue is (as the B cell, the T cell, histocyte or the like), central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, the tumour in particular tissues (as dental tissue or the like) source, for example, cancer of the stomach, liver cancer, large bowel cancer, mammary cancer, lung cancer, prostate cancer, cervical cancer, carcinoma of the pancreas, esophagus cancer or the like.

Polypeptide of the present invention still is a kind of immunomodulator, has immunological enhancement or immunosuppressive action.Polypeptide of the present invention can be used for the treatment of some diseases, and these diseases comprise immunoreactive anergy, or abnormal immune reaction, or the host defend invalid.Polypeptide of the present invention and its antibody also have effect to damage, defective or the imbalance class disease of immuning tissue, particularly for disease of hematopoietic system (as pernicious anemia), tetter (as psoriasis), autoimmune disease (as rheumatoid arthritis), radiation disease and immune lymphocytic generation and adjusting have extremely close relation.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check (antagonist) HABCT39's medicament.Agonist improves HABCT39's 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, the HABCT39 with mark cultivates with mammalian cell or the film preparation of expressing the HABCT39.Measure the medicine raising then or check this interactional ability.

HABCT39's antagonist comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.HABCT39's antagonist can combine and eliminate its function with the HABCT39, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the HABCT39 can be added during bioanalysis measures, by measuring compound interactional influence between HABCT39 and its acceptor is determined 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.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with HABCT39's bonded peptide molecule obtains.During screening, generally tackle HABCT39's molecule and carry out mark.

The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at the HABCT39 antigenic determinant.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 method of the available HABCT39's direct injection of the production of polyclonal antibody immune animal (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.Preparation HABCT39's the technology of monoclonal antibody include but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.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 anti-HABCT39's single-chain antibody.

Anti-HABCT39's antibody can be used in the immunohistochemistry technology, detects the HABCT39 in the biopsy specimen.

With the also available labelled with radioisotope of HABCT39's bonded monoclonal antibody, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of HABCT39's high-affinity 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 HABCT39's positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with the HABCT39.The antibody that gives suitable dosage can stimulate or block HABCT39's generation or activity.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization HABCT39 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The HABCT39's level that is detected in the test can be with laying down a definition the importance of HABCT39 in various diseases and be used to the disease of diagnosing the HABCT39 to work.

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 carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.

Coding HABCT39's polynucleotide also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to HABCT39's nothing expression or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the HABCT39 who expresses variation, to suppress endogenic HABCT39's activity.For example, a kind of HABCT39 of variation can be the HABCT39 who shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of HABCT39's expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for coding HABCT39's polynucleotide are transferred in the cell.The method of recombinant viral vector that structure carries coding HABCT39's polynucleotide is found in existing document (Sambrook, et al.).Reorganization coding HABCT39's polynucleotide can be packaged in the liposome and be transferred in the cell in addition.

Polynucleotide import 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.

Suppress the oligonucleotide (comprising sense-rna and DNA) of HABCT39 mRNA 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.

Coding HABCT39's polynucleotide can be used for the diagnosis with HABCT39's relative disease.The expression that coding HABCT39's polynucleotide can be used for detecting HABCT39 HABCT39's whether or under morbid state unconventionality expression.As the HABCT39's that encodes dna sequence dna can be used for biopsy specimen is hybridized to judge HABCT39's expression situation.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 the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect the HABCT39 with the special primer of HABCT39.

The sudden change that detects HABCT39's gene also can be used for the disease of diagnosing the HABCT39 to be correlated with.The form of HABCT39 sudden change comprises that the point mutation compared with normal wild type HABCT39 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.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, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch MedicalLibrary).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.

Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.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.The HABCT39 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's HABCT39 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the proteic amino acid sequence homology comparison diagram of the abc transport of HABCT39 of the present invention and fission yeast.The top sequence is the HABCT39, and the below sequence is the abc transport albumen of fission yeast.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is isolating HABCT39's polyacrylamide gel electrophoresis figure (SDS-PAGE).39kDa is proteinic molecular weight.The arrow indication is isolated protein band.

{ embodiment }

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:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: HABCT39's clone

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dye terminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 1027B04 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 1027B04 clone is 2049bp (shown in Seq ID NO:1), from 685bp to 1746bp the open reading frame (ORF) of a 1062bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-1027B04, encoded protein matter called after HABCT39.Embodiment 2:cDNA clone's homology retrieval

With HABCT39's of the present invention sequence and encoded protein sequence thereof, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with HABCT39's homology of the present invention is the abc transport albumen of a kind of known fission yeast, and its encoded protein number is AL022299 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 53%; Similarity is 71%.Embodiment 3: with RT-PCR method clones coding HABCT39's gene

Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:

Primer1：5’-GGGGCCGCCGTTACGGGCTGGTG-3’(SEQ?ID?NO：3)

Primer2：5’-CTATGTAAATTAAATCTCTCCCCG-3’(SEQ?ID?NO：4)

Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;

Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-aGtin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-2049bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed HABCT39's expression of gene:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is HABCT39's coding region sequence (685bp to 174 6bp) of pcr amplification shown in Figure 1.Probe (about 2 * 10 with the 32P-mark ⁶Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH ₂PO ₄(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.Embodiment 5: reorganization HABCT39's vivoexpression, separation and purifying

According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-CCCCATATGATGCTGGATGTCAGGGCCATCCTGT-3’(Seq?ID?No：5)

Primer4：5’-CATGGATCCCTAGAGGAAGCCTTCGCGGCGGAAC-3’(Seq?ID?No：6)

5 ' end of these two sections primers contains NdeI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NdeI and BamHI restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-1027B04 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-1027B04 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NdeI and BamHI, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-1027B04) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE 3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-1027B04) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein HABCT39 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 39kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 6 anti-HABCT39's production of antibodies

With the synthetic specific polypeptide of following HABCT39: the NH of Peptide synthesizer (PE company product) ₂-Met-Leu-Asp-Val-Arg-Ala-Ile-Leu-Trp-Leu-Glu-Asn-Tyr-Leu-Gln-COOH (SEQ ID NO:7).

Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with the HABCT39 specifically.

Sequence table (1) general information: (ii) denomination of invention: HABCT39 and encoding sequence thereof be the sequence number (iii): the information of 7 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 2049bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：1： 1 GGGGCCGCCGTTACGGGCTGGTGGGGCGGAATGGGTTGGGGAAGACAACGTTACTGAAGA 61 TGCTGGCCACCCGGAGTCTGCGGGTTCCAGCCCACATTTCCCTGCTGCACGTTGAGCAAG121 AGGTTGCTGGAGATGACACTCCTGCCCTGCAGAGTGTGCTGGAGAGTGACAGTGTGCGAG181 AGGATTTGCTACGGAGGGAGCGGGAGCTCACTGCCCAGATTGCTGCTGGCAGGGCGGAGG241 GCTCTGAAGCTGCAGAGCTGGCAGAAATCTATGCCAAACTGGAGGAGATTGAGGCTGACA301 AGGCACCTGCCAGGGCATCAGTCATTCTCGCTGGGCTTGGCTTTACCCCTAAAATGCAGC361 AGCAGCCCACCCGGGAGTTCTCAGGTGGCTGGAGGATGAGGCTGGCCCTGGCCCGGGCCC421 TCTTTGCTAGGTGAGTCTCCTGGGCCAGTGTATGAAGCCCTATGGAAGATTGGTCTTAGC481 TCTTGGGAGGTGGCATGAGTTGCTGACCTAAAACCTCAGGCCATGTATTGTTTTTCATAG541 GCCAGATCTTCTGCTGTTAGATGGTGAGTTTGAAATGGGGCACCCTGACTTTAGGATGGG601 CAGGGGTGGTAGCATCCAAGTTCCTAGTTCTCCAGCCCTTCAATCCCAAGTGTGTGCTCC661 CCTGCTTCTCCAGAACCTACAAACATGCTGGATGTCAGGGCCATCCTGTGGCTGGAGAAT721 TACCTGCAGACGTGGCCCTCCACCATCCTAGTCGTCTCCCACGACCGCAACTTCTTGAAT781 GCCATCGCCACAGACATCATCCACCTGCACAGCCAGCGGCTAGATGGTTACCGGGGAGAC841 TTTGAGACCTTCATCAAGAGTAAGCAGGAGCGGCTGCTCAACCAGCAGCGTGAATATGAG901 GCGCAGCAGCAGTATCGCCAGCACATCCAGGTTTTCATTGACCGGTTTCGCTACAATGCC961 AACAGGGCCTCTCAAGTGCAGAGTAAACTCAAGATGCTGGAGAAGCTGCCTGAGCTGAAG1021 CCTGTGGACAAGGAATCAGAGGTCGTAATGAAGTTCCCTGATGGGTTTGAGAAGTTCTCG1081 CCGCCAATTCTGCAGCTAGATGAGGTGGATTTCTACTACGATCCGAAGCACGTCATCTTC1141 AGTCGCCTCTCTGTGTCTGCTGATCTCGAGTCTCGCATCTGTGTGGTTGGAGAGAATGGG1201 GCTGGGAAGTCTACCATGCTGAAGCTGCTTTTGGGGGACCTGGCACCTGTTCGGGGCATC1261 AGACACGCTCACAGGAATCTGAAGATTGGCTATTTCAGCCAGCACCATGTGGAGCAGCTG1321 GACCTAAACGTCAGTGCTGTGGAACTGCTGGCACGCAAGTTTCCTGGGCGGCCTGAGGAG1381 GAGTACCGTCACCAGCTGGGTCGGTATGGCATCTCCGGAGAACTGGCCATGCGTCCTCTT1441 GCCAGCCTGTCTGGGGGCCAGAAGAGCCGAGTGGCCTTTGCTCAGATGACTATGCCCTGC1501 CCCAACTTCTACATTCTGGATGAACCCACAAACCACCTGGACATGGAGACCATTGAGGCT1561 CTGGGCCGTGCCCTCAACAATTTCAGGGGTGGTGTGATTCTGGTGTCCCACGATGAGCGC1621 TTTATCAGGCTGGTGTGCCGGGAGTTGTGGGTATGCGAAGGAGGCGGCGTCACCCGTGTG1681 GAAGGAGGATTTGACCAGTACCGCGCCCTCCTCCAGGAACAGTTCCGCCGCGAAGGCTTC1741 CTCTAGGGCCACCAGGCTGAGGACTCGCCCAGGACATGGACTGGTCTCTCAGACCCCTGG1801 GCCACCATGTAGGCCACCACTCCAGGCCGTGGACTTCCCCCAACTTGGGGACAGCCTTAT1861 TCCCAAATGTCTCTATCCTTTTGACTGGAGCATCTTCTGCACAACCTTGGGAGCCCATCC1921 AAGGGTTGGTGAGGACTGGTCTCCCGGGGGTGGGGGTCTGGGGGGTACCCTCTGGGGTTA1981 TAGATTCCCCCACTGCCCCAGCTCTGACTGGACCCCAAGTGGCTGCTATGTAAATTAAAT2041 CTCTCCCCG

(3) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 353 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO：2： 1 Met Leu Asp Val Arg Ala Ile Leu Trp Leu Glu Asn Tyr Leu Gln16 Thr Trp Pro Ser Thr Ile Leu Val Val Ser His Asp Arg Asn Phe31 Leu Asn Ala Ile Ala Thr Asp Ile Ile His Leu His Ser Gln Arg46 Leu Asp Gly Tyr Arg Gly Asp Phe Glu Thr Phe Ile Lys Ser Lys61 Gln Glu Arg Leu Leu Asn Gln Gln Arg Glu Tyr Glu Ala Gln Gln76 Gln Tyr Arg Gln His Ile Gln Val Phe Ile Asp Arg Phe Arg Tyr 91 Asn Ala Asn Arg Ala Ser Gln Val Gln Ser Lys Leu Lys Met Leu106 Glu Lys Leu Pro Glu Leu Lys Pro Val Asp Lys Glu Ser Glu Val121 Val Met Lys Phe Pro Asp Gly Phe Glu Lys Phe Ser Pro Pro Ile136 Leu Gln Leu Asp Glu Val Asp Phe Tyr Tyr Asp Pro Lys His Val151 Ile Phe Ser Arg Leu Ser Val Ser Ala Asp Leu Glu Ser Arg Ile166 Cys Val Val Gly Glu Asn Gly Ala Gly Lys Ser Thr Met Leu Lys181 Leu Leu Leu Gly Asp Leu Ala Pro Val Arg Gly Ile Arg His Ala196 His Arg Asn Leu Lys Ile Gly Tyr Phe Ser Gln His His Val Glu211 Gln Leu Asp Leu Asn Val Ser Ala Val Glu Leu Leu Ala Arg Lys226 Phe Pro Gly Arg Pro Glu Glu Glu Tyr Arg His Gln Leu Gly Arg241 Tyr Gly Ile Ser Gly Glu Leu Ala Met Arg Pro Leu Ala Ser Leu256 Ser Gly Gly Gln Lys Ser Arg Val Ala Phe Ala Gln Met Thr Met271 Pro Cys Pro Asn Phe Tyr Ile Leu Asp Glu Pro Thr Asn His Leu286 Asp Met Glu Thr Ile Glu Ala Leu Gly Arg Ala Leu Asn Asn Phe301 Arg Gly Gly Val Ile Leu Val Ser His Asp Glu Arg Phe Ile Arg316 Leu Val Cys Arg Glu Leu Trp Val Cys Glu Gly Gly Gly Val Thr331 Arg Val Glu Gly Gly Phe Asp Gln Tyr Arg Ala Leu Leu Gln Glu346 Gln Phe Arg Arg Glu Gly Phe Leu

(4) information of SEQ ID NO:3

(i) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: information (i) sequence signature of SEQ ID NO:3:GGGGCCGCCGTTACGGGCTGGTG 23 (5) SEQ ID NO:4

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:4:CTATGTAAATTAAATCTCTCCCCG 24 (6) SEQ ID NO:5

(A) length: 32 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:5:CAGCCATGGCGGGGAAGAAGAATGTTCTGTCG 32 (7) SEQ ID NO:6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:6:CCCGGATCCCGCTGCTTGGCCTTCTTCAC 29 (8) SEQ ID NO:7: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): polypeptide (xi) sequence description: SEQ ID NO:7:Met-Ala-Gly-Lys-Lys-Asn-Val-Leu-Ser-Ser-Leu-Ala-Val-Tyr-Ala 15

Claims (18)

1, a kind of isolated polypeptide-HABCT39 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.

4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding have aminoacid sequence shown in the SEQ ID NO:2 polypeptide or its fragment, analogue, derive

The polynucleotide of thing;

(b) with polynucleotide (a) complementary polynucleotide; Or

(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise coding and have SEQ

The polynucleotide of aminoacid sequence shown in the ID NO:2.

6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-2049 position among the sequence of 685-1746 position among the SEQ IDNO:1 or the SEQ ID NO:1.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:

(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or

(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.

9, a kind of preparation method with the active polypeptide of HABCT39 is characterized in that described method comprises:

(a) expressing under HABCT39's condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of HABCT39.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with HABCT39's specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses the HABCT39.

12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate the HABCT39 in vivo, the method for external activity.

14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen HABCT39's stand-in, agonist, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

17,, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with the HABCT39 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.