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CN1292385A - New polypeptide-human DNA-PK interaction protein 75 and polynucleotide coding this polypeptide - Google Patents

New polypeptide-human DNA-PK interaction protein 75 and polynucleotide coding this polypeptide Download PDF

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CN1292385A
CN1292385A CN 99116950 CN99116950A CN1292385A CN 1292385 A CN1292385 A CN 1292385A CN 99116950 CN99116950 CN 99116950 CN 99116950 A CN99116950 A CN 99116950A CN 1292385 A CN1292385 A CN 1292385A
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polypeptide
polynucleotide
human dna
interaction protein
dna
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毛裕民
谢毅
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Shanghai Bodao Gene Technology Co Ltd
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Shanghai Bodao Gene Technology Co Ltd
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Abstract

The present invention discloses a new polypeptide-human DNA-PK interaction protein 75, polynucleotide coding said polypeptide and method for producing this polypeptide by means of recombination technology. Said invention also discloses the method for curing several diseases, such as malignant tumor, blood disease, HIV infection, immune disease and various inflammation by using said polypeptide. Said invention also discloses an antagonist for resisting said polypeptide and its therapeutic effect. It also discloses the application of polynucleotide for coding this new human DNA-PK interaction protein 75.

Description

The polynucleotide of a kind of new polypeptide--human DNA-PK interaction protein 75 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide--human DNA-PK interaction protein 75, 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.
DNA dependent kinases (DNA-PK) is unique by the terminal activatory eukaryote of DNA kinases.Biochemical research discloses, and the activation of DNA dependent kinases (DNA-PK) depends on the terminal protein-bonded stimulation of DNA.(Mutat?Res?1997?℃t；385(1)：13-20)
DNA dependent kinases (DNA-PK) is a kind ofly to be made up of a big catalytic subunit and Ku heterodimer, and the Ku heterodimer is by regulating kinase whose activity with combining of DNA.(Bi℃himie?1999?Jan-Feb；81(1-2)：117-25)
The mechanism that DNA dependent kinases (DNA-PK) is regulated by DNA has been studied clear at present, and result of experiment shows that DNA-PK is via some independences but interactional site with strand or double-stranded combining.(EMBO?J?1999?Mar?1；18(5)：1114-23)
The catalytic subunit of DNA dependent kinases (DNA-PK) is to belong to phosphoric acid acyl inositol 3-kinase family, nearest genetics and biochemical studies show that, DNA-PK has effect in processes such as the injury repairing of immunoglobulin (Ig)/TXi Baoshouti gene composite, double-stranded DNA, autoimmunization.(Curr?Opin?Cell?Biol1996?Jun；8(3)：325-30)
DNA dependent kinases (DNA-PK) is a kind of important dna damage reparative factor, can be very responsive to radiation if cell lacks DNA-PK.DNA dependent kinases (DNA-PK) is also relevant with lymphocytic reparation, as its shortage, then can cause immune deficiency dominance.(Bi℃himie?1999?Jan-Feb；81(1-2)：117-25)
Deep discovers, has a kind of albumen and DNA-PK that extremely strong interaction is arranged, and this albumen and Phosphoric acid esterase have certain homology, is called as DNA-PK interaction protein (KIP).It is relevant with calcium ion-binding protein family that KIP is inferred, KIP and the interaction of DNA-PK are the terminal adjusting factors that is connected of DNA, and the interaction of this and kinases and Phosphoric acid esterase has similarity.(Mutat?Res?1997?℃t；385(1)：13-20)
The result of RT-PCR shows that KIP is wide expression in a lot of people's tissues.According to radiation hybridization location and the localized result of fluorescent hybridization, the polynucleotide of encoded K IP are located in the q24 zone of human chromosome group 15.(Bi℃him?Biophys?Acta?1999?Jan?18；1444(1)：143-7)
Polypeptide of the present invention is accredited as human DNA-PK interaction protein 75 (HKIP75) by deduction, and this is amino acid homology result relatively.
An object of the present invention is to provide isolating new polypeptide--human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 of encoding.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the human DNA-PK interaction protein 75 of encoding.
Another object of the present invention provides the method for producing human DNA-PK interaction protein 75.
Another object of the present invention provides at polypeptide of the present invention--the antibody of human DNA-PK interaction protein 75.
Another object of the present invention has provided at polypeptide of the present invention--the simulated compound of human DNA-PK interaction protein 75, antagonist, agonist, inhibitor.
Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with human DNA-PK interaction protein 75.
In a first aspect of the present invention, novel isolated human DNA-PK interaction protein 75 is provided, this polypeptide is the people source, and it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned human DNA-PK interaction protein 75 of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 5-2062 position among the SEQ ID NO:1; (b) has the sequence of 1-2587 position among the SEQ ID NO:1.
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.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
As used herein, " 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 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 human DNA-PK interaction protein 75 " is meant that human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of human DNA-PK interaction protein 75 polypeptide can be used amino acid sequence analysis.
The invention provides a kind of new polypeptide--human DNA-PK interaction protein 75, it is made up of the aminoacid sequence shown in the SEQID NO: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 fragment, derivative and the analogue of human DNA-PK interaction protein 75.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of human DNA-PK interaction protein 75 of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is 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 2587 bases, and its open reading frame (5-2062) 685 amino acid of having encoded find relatively that according to amino acid sequence homologous this polypeptide and zymic KIP have 58% homology.
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 the polynucleotide of coding human DNA-PK interaction protein 75.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding human DNA-PK interaction protein 75 of the present invention 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 the transcript of mensuration human DNA-PK interaction protein 75; (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 human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology through the genetically engineered generation.
Among the present invention, the polynucleotide sequence of coding human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 of encoding 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 P of lambda particles phage LPromotor; 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, the polynucleotide of coding human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people human DNA-PK interaction protein 75, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(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.
Because polypeptide HKIP75 of the present invention and DNA-PK have extremely strong interaction, it is the terminal adjusting factor that is connected of DNA with the interaction of DNA-PK, thereby HKIP75 has important effect in processes such as the injury repairing of immunoglobulin (Ig)/TXi Baoshouti gene composite, double-stranded DNA, autoimmunization.
HKIP75 can be used for the diagnosis and the treatment of a lot of diseases of people, as malignant tumour, and immunological disease, people's acquired immune deficiency syndrome (AIDS), endocrine system disease, nervous system disorders or the like.
Polypeptide of the present invention can be used for treating people's malignant tumour, includes but not limited to cancer of the stomach, liver cancer, large bowel cancer, mammary cancer, lung cancer, prostate cancer, cervical cancer, carcinoma of the pancreas, esophagus cancer.
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) human DNA-PK interaction protein 75.Agonist improves human DNA-PK interaction protein 75 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 in the presence of medicine, the film preparation of mammalian cell or expression human DNA-PK interaction protein 75 be cultivated with the human DNA-PK interaction protein 75 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of human DNA-PK interaction protein 75 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of human DNA-PK interaction protein 75 can combine and eliminate its function with human DNA-PK interaction protein 75, 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, human DNA-PK interaction protein 75 can be added in the bioanalysis mensuration, interactional influence between human DNA-PK interaction protein 75 and its acceptor be determined whether compound is antagonist by measuring compound.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 human DNA-PK interaction protein 75 bonded peptide molecule obtains.During screening, generally tackle the human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 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.The technology of the monoclonal antibody of preparation human DNA-PK interaction protein 75 includes 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 the single-chain antibody of anti-human DNA-PK interaction protein 75.
The antibody of anti-human DNA-PK interaction protein 75 can be used in the immunohistochemistry technology, detects the human DNA-PK interaction protein 75 in the biopsy specimen.
With the also available labelled with radioisotope of human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 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 the human DNA-PK interaction protein 75 positive cells.
The disease that antibody among the present invention can be used for treating or prevention is relevant with human DNA-PK interaction protein 75.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human DNA-PK interaction protein 75.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization human DNA-PK interaction protein 75 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The human DNA-PK interaction protein 75 level that is detected in the test can be with laying down a definition the importance of human DNA-PK interaction protein 75 in various diseases and be used to the disease of diagnosing human DNA-PK interaction protein 75 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.
The polynucleotide of coding human DNA-PK interaction protein 75 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 the nothing expression of human DNA-PK interaction protein 75 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the human DNA-PK interaction protein 75 of expressing variation, to suppress endogenic human DNA-PK interaction protein 75 activity.For example, a kind of human DNA-PK interaction protein 75 of variation can be the human DNA-PK interaction protein 75 that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of human DNA-PK interaction protein 75 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 the polynucleotide of coding human DNA-PK interaction protein 75 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding human DNA-PK interaction protein 75 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 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.
The polynucleotide of coding human DNA-PK interaction protein 75 can be used for the diagnosis with the relative disease of human DNA-PK interaction protein 75.The unconventionality expression of the expression that the polynucleotide of coding human DNA-PK interaction protein 75 can be used for detecting human DNA-PK interaction protein 75 human DNA-PK interaction protein 75 whether or under morbid state.As the dna sequence dna of the human DNA-PK interaction protein 75 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of human DNA-PK interaction protein 75.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 human DNA-PK interaction protein 75 with the special primer of human DNA-PK interaction protein 75.
The sudden change that detects the human DNA-PK interaction protein 75 gene also can be used for the disease of diagnosing human DNA-PK interaction protein 75 relevant.The form of human DNA-PK interaction protein 75 sudden change comprises that the point mutation compared with normal wild type human DNA-PK interaction protein 75 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 Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
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.Human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75 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 amino acid sequence homology comparison diagram of human DNA-PK interaction protein 75 of the present invention and zymic KIP.The top sequence is a human DNA-PK interaction protein 75, and the below sequence is zymic KIP.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".
Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating human DNA-PK interaction protein.75kDa 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: the clone of human DNA-PK interaction protein 75
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 Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI377 automatic sequencing view (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 0768D03 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 0768D03 clone is 2587bp (shown in Seq ID NO:1), from 5bp to 2062bp the open reading frame (ORF) of a 2058bp, the new protein (shown in SeqID NO:2) of encoding arranged.We are with this clone's called after pBS-0768D03, encoded protein matter called after human DNA-PK interaction protein 75.
Embodiment 2:cDNA clone's homology retrieval
With the sequence and the encoded protein sequence thereof of human DNA-PK interaction protein 75 of the present invention, with Blast program (Basic
Local 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 human DNA-PK interaction protein 75 homology of the present invention is a kind of known yeast KIP gene, and its encoded protein number is Z73098 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 34%; Similarity is 62%.Embodiment 3: with the gene of RT-PCR method clones coding human DNA-PK interaction protein 75
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’-GTTAATGGCCACTGTACACCAGACT-3’(SEQ?ID?NO：3)
Primer2：5’-AGCAATCAAAAGATGTTTATTTTT-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-actin 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 1-2587bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed the human DNA-PK interaction protein 75 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 gRNA, 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 the human DNA-PK interaction protein 75 coding region sequence (5bp to 2062bp) 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: vivoexpression, separation and the purifying of recombinant human dna-PK interaction protein 75
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’-CCCGGATCCATGGCCACTGTACACCAGAC-3’(Seq?ID?No：5)
Primer4：5’-CCCCTCGAGTCACCTCTTCTGCTCCCAAA-3’(Seq?ID?No：6)
5 ' end of these two sections primers contains BamH I and Xho I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, BamH I and Xho I 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-0768D03 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0768D03 plasmid 1Opg, 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 BamH I and Xho I, 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-0768D03) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0768D03) 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 human DNA-PK interaction protein 75 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 75kDa 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-human DNA-PK interaction protein 75 production of antibodies
Synthesize the specific polypeptide of following human DNA-PK interaction protein 75 with Peptide synthesizer (PE company product):
NH2-Met-Ala-Thr-Val-His-Gln-Thr-Gln-Pro-Thr-Val-Gln-Val-Leu-Leu-OH(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 people DHA-PK interaction protein 75 specifically.
Sequence Table (A) General information: (Ⅱ) Title of Invention: Human DNA-PK-interacting protein 75 (Ⅲ) Serial Number: 7 (2) SEQ ID NO: 1 of the message: (Ⅰ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2587bp (B) TYPE: nucleic acid (C) chain resistance: double-stranded (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: cDNA (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 1 GTTAATGGCCACTGTACACCAGACTCAGCCTACAGTACAGGTGTTATTATCTACTTCTGA 61 ATTTGTTGGA GCATTGGACTTAATAGCAACAACACAAGAGGTTCTACAGCAGGAACTTCA 121 GGGCATTCAC AGTTTCCGGCATTTGGGATCACAGCTTTGTGAATTAGAAAAACTGATAGA 181 TAAAATGATG ATTGCAGAATTTTCTACTTATTCTCACAGTGACTTAAATAGACCACTGGA 241 AGATGACTGT CAAGTTTTAGAAGAGGAAAGACTAATATCTCTTGTATTTGGACTTTTAAA 301 ACAAAGAAAG CTTAATTTTTTAGAAATCTATGGTGAAAAAATGGTTATTACAGCAAAGAA 361 TATCATTAAA CAGTGTGTGATTAATAAAGTTTCACAAACAGAAGAAATAGACACAGATGT 421 TGTTGTGAAG CTTGCAGATCAGATGAGAATGTTGAATTTTCCCCAGTGGTTTGATCTGCT 481 CAAGGATATT TTCTCTAAGTTTACAATTTTCCTACAGAGAGTGAAGGCAACATTAAATAT 541 CATTCACAGT GTTGTTCTCTCAGTTCTTGACAAAAACCAAAGGACTAGAGAATTGGAAGA 601 GATTTCACAA CAGAAGAATGCTGCAAAAGATAATTCACTGGACACAGAGGTGGCTTATTT 661 AATCCATGAA GGCATGTTTATAAGTGATGCATTCGGTGAGGGTGAGCTAACACCTATAGC 721 AGTTGACACT ACCTCTCAAAGAAATGCATCTCCAAATAGTGAGCCCTGCAGCAGTGATTC 781 TGTATCCGAG CCAGAATGTACTACTGATTCTTCATCCAGCAAAGAGCACACATCATCATC 841 TGCTATTCCA GGAGGTGTGGATATTATGGTCAGTGAAGATATGAAATTAACTGACTCAGA 901 GCTAGGAAAG CTGGCAAATAATATCCAGGAATTATTATATAGTGCCTCAGATATATGCCA 961 TGATCGAGCT GTCAAATTTCTCATGTCAAGAGCAAAGGATGGTTTTCTTGAGAAGCTAAA 1021 TTCCATGGAATTCATAACACTTTCTAGATTAATGGAAACATTCATTTTAGACACCGAACA 1081 GATCTGTGGAAGAAAAAGCACGTCATTACTTGGAGCACTTCAGAGCCAAGCTATTAAGTT 1141 TGTAAATAGGTTTCATGAAGAGAGAAAAACCAAGCTCAGCCTCCTCTTAGACAATGAGCG 1201 CTGGAAGCAAGCAGATGTTCCTGCAGAATTTCAGGATCTTGTTGATTCTCTGTCAGATGG 1261 GAAGATTGCTTTACCTGAAAAAAAATCAGGAGCCACAGAAGAAAGGAAACCAGCTGAAGT 1321 TCTTATTGTCGAGGGACAACAGTATGCAGTTGTTGGAACCGTATTGCTGTTAATAAGAAT 1381 TATCCTTGAATATTGCCAGTGTGTGGATAACATCCCATCTGTTACTACTGACATGCTTAC 1441 TCGTCTGTCAGATTTATTGAAGTACTTCAATTCAAGAAGTTGCCAGTTAGTTCTTGGAGC 1501 TGGTGCACTGCAAGTTGTTGGACTAAAAACGATAACTACAAAAAATTTGGCTCTTTCTTC 1561 ACGATGTTTGCAGTTAATTGTGCACTACATTCCTGTGATCCGGGCTCATTTTGAAGCTCG 1621 ACTACCACCTAAGCAATATAGCATGCTTAGGCATTTTGATCATATCACTAAGGACTACCA 1681 TGATCACATAGCTGAAATATCAGCTAAGCTTGTAGCGATAATGGATAGCTTATTTGACAA 1741 GCTGTTATCTAAGTATGAAGTGAAGGCTCCTGTTCCTTCTGCCTGTTTCAGGAATATTTG 1801 TAAGCAAATGACAAAAATGCACGAAGCTATATTTGATCTCCTTCCAGAAGAACAAACACA 1861 GATGTTATTTTTAAGAATTAATGCAAGTTATAAACTCCACTTGAAAAAGCAGTTATCTCA 1921 CTTAAATGTGATAAATGATGGAGGACCTCAAAATGGGTTGGTCACAGCAGATGTAGCTTT 1981 TTACACTGGAAATCTTCAAGCCTTAAAAGGCCTTAAAGATTTGGACCTAAATATGGCCGA 2041 AATTTGGGAGCAGAAGAGGTGATGTCATCCTGGAAAACTGGGTAGTTCATCTGACCATGG 2101 GATGTGTTTGTTATGAAGAAAATCTGGATGCCTGTGATTCGAGAATTGAACCTGAAACCC 2161 AAAGTGAACTGGGGTGGGGGAAGGGAAAAAGGAAAGTATCAAGTGTTGGGAAACTGGATT 2221 CAGTGGGATCTACAAGGAATGTCATTTTTGTGCATCCTACAGTGAGGAGTAACTGATCAG 2281 GTGTCTATAACATTTTTCATTCTCTCTGGAAACAGACTCAGGTTTCTTTGGACCAAATCC 2341 AAAAGAACACATAGCTGTAACACAGCTGTAGTTGACTAGAATGCTCTGTATACTTTATAT 2401 TAAAAAATGCTTTGCATTTCTTCCAGTGCAATGAAATTCATATGGTGTCCCACCTTATTT 2461 AATGATGGTACAATTTAAAATCTTAGTCAACTTCTGTAGAAAGTTTTCTCTATGAAAGTA 2521 AAGCTGTTTGAAAAATTATTATTTTTTTACAGATCTTTCTATAAAAAATAAACATCTTTT 2581 GATTGCT (3) SEQ ID NO: 2 of the message: (Ⅰ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 685 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: peptide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 1 Met Ala Thr Val His Gln Thr Gln Pro Thr Val Gln Val Leu Leu 16 Ser Thr Ser Glu Phe Val Gly Ala Leu Asp Leu Ile Ala Thr Thr 31 Gln Glu Val Leu Gln Gln Glu Leu Gln Gly Ile His Ser Phe Arg 46 His Leu Gly Ser Gln Leu Cys Glu Leu Glu Lys Leu Ile Asp Lys 61 Met Met Ile Ala Glu Phe Ser Thr Tyr Ser His Ser Asp Leu Asn 76 Arg Pro Leu Glu Asp Asp Cys Gln Val Leu Glu Glu Glu Arg Leu 91 Ile Ser Leu Val Phe Gly Leu Leu Lys Gln Arg Lys Leu Asn Phe 106 Leu Glu Ile Tyr Gly Glu Lys Met Val Ile Thr Ala Lys Asn Ile 121 Ile Lys Gln Cys Val Ile Asn Lys Val Ser Gln Thr Glu Glu Ile 136 Asp Thr Asp Val Val Val Lys Leu Ala Asp Gln Met Arg Met Leu 151 Asn Phe Pro Gln Trp Phe Asp Leu Leu Lys Asp Ile Phe Ser Lys 166 Phe Thr Ile Phe Leu Gln Arg Val Lys Ala Thr Leu Asn Ile Ile 181 His Ser Val Val Leu Ser Val Leu Asp Lys Asn Gln Arg Thr Arg 196 Glu Leu Glu Glu Ile Ser Gln Gln Lys Asn Ala Ala Lys Asp Asn 211 Ser Leu Asp Thr Glu Val Ala Tyr Leu Ile His Glu Gly Met Phe 226 Ile Ser Asp Ala Phe Gly Glu Gly Glu Leu Thr Pro Ile Ala Val 241 Asp Thr Thr Ser Gln Arg Asn Ala Ser Pro Asn Ser Glu Pro Cys 256 Ser Ser Asp Ser Val Ser Glu Pro Glu Cys Thr Thr Asp Ser Ser 271 Ser Ser Lys Glu His Thr Ser Ser Ser Ala Ile Pro Gly Gly Val 286 Asp Ile Met Val Ser Glu Asp Met Lys Leu Thr Asp Ser Glu Leu 301 Gly Lys Leu Ala Asn Asn Ile Gln Glu Leu Leu Tyr Ser Ala Ser 316 Asp Ile Cys His Asp Arg Ala Val Lys Phe Leu Met Ser Arg Ala 331 Lys Asp Gly Phe Leu Glu Lys Leu Asn Ser Met Glu Phe Ile Thr 346 Leu Ser Arg Leu Met Glu Thr Phe Ile Leu Asp Thr Glu Gln Ile 361 Cys Gly Arg Lys Ser Thr Ser Leu Leu Gly Ala Leu Gln Ser Gln 376 Ala Ile Lys Phe Val Asn Arg Phe His Glu Glu Arg Lys Thr Lys 391 Leu Ser Leu Leu Leu Asp Asn Glu Arg Trp Lys Gln Ala Asp Val 406 Pro Ala Glu Phe Gln Asp Leu Val Asp Ser Leu Ser Asp Gly Lys 421 Ile Ala Leu Pro Glu Lys Lys Ser Gly Ala Thr Glu Glu Arg Lys 436 Pro Ala Glu Val Leu Ile Val Glu Gly Gln Gln Tyr Ala Val Val 451 Gly Thr Val Leu Leu Leu Ile Arg Ile Ile Leu Glu Tyr Cys Gln 466 Cys Val Asp Asn Ile Pro Ser Val Thr Thr Asp Met Leu Thr Arg 481 Leu Ser Asp Leu Leu Lys Tyr Phe Asn Ser Arg Ser Cys Gln Leu 496 Val Leu Gly Ala Gly Ala Leu Gln Val Val Gly Leu Lys Thr Ile 511 Thr Thr Lys Asn Leu Ala Leu Ser Ser Arg Cys Leu Gln Leu Ile 526 Val His Tyr Ile Pro Val Ile Arg Ala His Phe Glu Ala Arg Leu 541 Pro Pro Lys Gln Tyr Ser Met Leu Arg His Phe Asp His Ile Thr 556 Lys Asp Tyr His Asp His Ile Ala Glu Ile Ser Ala Lys Leu Val 571 Ala Ile Met Asp Ser Leu Phe Asp Lys Leu Leu Ser Lys Tyr Glu 586 Val Lys Ala Pro Val Pro Ser Ala Cys Phe Arg Asn Ile Cys Lys 601 Gln Met Thr Lys Met His Glu Ala Ile Phe Asp Leu Leu Pro Glu 616 Glu Gln Thr Gln Met Leu Phe Leu Arg Ile Asn Ala Ser Tyr Lys 631 Leu His Leu Lys Lys Gln Leu Ser His Leu Asn Val Ile Asn Asp 646 Gly Gly Pro Gln Asn Gly Leu Val Thr Ala Asp Val Ala Phe Tyr 661 Thr Gly Asn Leu Gln Ala Leu Lys Gly Leu Lys Asp Leu Asp Leu 676 Asn Met Ala Glu Ile Trp Glu Gln Lys Arg (4) SEQ ID NO: 3 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 25 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTTAATGGCCACTGTACACCAGACT 25 (5) SEQ ID NO: 4 for information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 24 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AGCAATCAAAAGATGTTTATTTTT 24 (6) SEQ ID NO: 5 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCCGGATCCATGGCCACTGTACACCAGAC 29 (7) SEQ ID NO: 6 Information (Ⅰ) SEQUENCE CHARACTERISTICS (A) LENGTH: 29 bases (B) TYPE: nucleic acid (C) chain: single-chain (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: oligonucleotide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCCCTCGAGTCACCTCTTCTGCTCCCAAA 29 8) SEQ ID NO: 7 information about: (Ⅰ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (Ⅱ) MOLECULE TYPE: peptide (Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 7: NH2-Met-Ala-Thr-Val-His-Gln-Thr-Gln-Pro-Thr-Val-Gln-Val-Leu-Leu-OH ...

Claims (18)

1, a kind of isolated polypeptide-human DNA-PK interaction protein 75 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ IDNO:2 or the active fragments of its polypeptide, analogue or derivative.
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 has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;
(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 the polynucleotide that coding has aminoacid sequence shown in the SEQID NO:2.
6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-2587 position among the sequence of 5-2062 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 arbitrary claim that require among the 4-6.
9, a kind of preparation method with the active polypeptide of human DNA-PK interaction protein 75 is characterized in that described method comprises:
(a) expressing under the human DNA-PK interaction protein 75 condition, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have the active polypeptide of human DNA-PK interaction protein 75.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with human DNA-PK interaction protein 75 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 human DNA-PK interaction protein 75.
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 human DNA-PK interaction protein 75 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 the stand-in of human DNA-PK interaction protein 75, 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 human DNA-PK interaction protein 75 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.
CN 99116950 1999-10-10 1999-10-10 New polypeptide-human DNA-PK interaction protein 75 and polynucleotide coding this polypeptide Pending CN1292385A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072830A3 (en) * 2001-02-09 2003-09-12 Incyte Genomics Inc Proteins associated with cell growth, differentiation, and death

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072830A3 (en) * 2001-02-09 2003-09-12 Incyte Genomics Inc Proteins associated with cell growth, differentiation, and death

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