CN1519320A - Polypeptide-human formamido pyrimidine-glycosylase 11.77 and polynucleotide for encoding polypoptide - Google Patents
Polypeptide-human formamido pyrimidine-glycosylase 11.77 and polynucleotide for encoding polypoptide Download PDFInfo
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- CN1519320A CN1519320A CNA031150772A CN03115077A CN1519320A CN 1519320 A CN1519320 A CN 1519320A CN A031150772 A CNA031150772 A CN A031150772A CN 03115077 A CN03115077 A CN 03115077A CN 1519320 A CN1519320 A CN 1519320A
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- polynucleotide
- polypeptide
- formamidopyrimidine
- dna
- dna glycosylase
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Abstract
A novel polypeptide-human formamidopyrimidine-DNA glycosylase 11.77, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating diseases, such as cancer, HIV infection, immunopathy, etc. the antagon of said polypeptide and its medical action, and the application of said polynucleotide are disclosed.
Description
Technical field
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---formamidopyrimidine-DNA glycosylase 11.77, 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.
Background technology
DNA is the carrier of genetic information, and it needs high fidelity of reproduction, and this not only depends on the perfect of the system of duplicating, and can correct the such class reparation of already present mistake system but also need.In evolution of long period of time, organism develop the system that can correct accidental misreplication and can the repairing environment factor and body in the system of damage of the dna molecular that causes of chemical substance.In dna molecular, except uridylic can infiltrate when the dna replication dna, cytosine(Cyt) also can be oxidized into uridylic by spontaneous deamination.VITAMIN B4 also can the deamination oxidation generate xanthoglobulin.In addition, also have other base derivative can be present in the DNA chain, for example 2,6-diamino-4-hydroxyl-5N-methyl formamido group pyrimidine (Fapy) and 7,8-dihydro-8-oxo guanines (8-OxoG) etc. have all been found its corresponding D NA glycosylase.The DNA glycosylase extensively exist with nearly all organism in.Whether have intrinsic AP endonuclease activity according to them, the DNA glycosylase can be divided into two classes: do not have AP endonuclease activity and a tool AP endonuclease activity.
Formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) belongs to second kind of DNA glycosylase, it is the enzyme that a kind of DNA of participation repairs, thereby its excision oxidation purine bases discharges 2,6-diamino-4-hydroxyl-5N-methyl formamido group pyrimidine (Fapy) and 7,8-dihydro-8-oxo guanine (8-OxoG) residue.FPG can also cut DNA in five purine pyrimidine sites (AP site) except having the glycosylase activity.FPG is a kind of monomeric protein, and molecular weight is about 32Kd, and its activity form requires to combine with zinc.The zinc binding site is positioned at proteic C-end parts, wherein contain four conservative and important halfcystines, be the zinc part, the conservative characteristic sequence template of this section is: C-x (2,4)-C-x-[GTAQ]-x-[IV]-x (7)-R-[GSTAN]-[STA]-x-[FYI]-C-x (2)-C-Q.Formamidopyrimidine-DNA glycosylase is very important for the existence of organism.If the gene of these enzymes is undergone mutation, people's physical appearance will be very poor, can catch diseases such as various cancerous swellings.The repair mechanism of formamidopyrimidine-DNA glycosylase is identical with the uridylic glycosylase.
Analysis by gene chip is found, at normal brain activity, liver cancer, muscle, tire brain, tire kidney, in tire lung, tire liver, Tiroidina, thymus gland, adult liver, lung, the glioma, polypeptide expression spectrum of the present invention is very approximate with the express spectra of formamidopyrimidine-DNA glycosylase, so the two function also may be similar.The present invention is named as formamidopyrimidine-DNA glycosylase 11.77.
Because formamidopyrimidine-DNA glycosylase 11.77 albumen play 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 formamidopyrimidine-DNA glycosylase 11.77 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new formamidopyrimidine-DNA glycosylase 11.77 protein coding genes also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of 1 sick diagnosis of exploitation disease and/or curative, and it is very important therefore separating its coding DNA.
Summary of the invention
An object of the present invention is to provide isolating new polypeptide-human formamidopyrimidine-DNA glycosylase 11.77 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 coding people formamidopyrimidine-DNA glycosylase 11.77.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding people formamidopyrimidine-DNA glycosylase 11.77.
Another object of the present invention provides the method for producing people's formamidopyrimidine-DNA glycosylase 11.77.
Another object of the present invention provides the antibody at polypeptide-human formamidopyrimidine-DNA glycosylase 11.77 of the present invention.
The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: have＜polypeptide or its examples of conservative variations, bioactive fragment or the derivative of 210〉2 aminoacid sequences.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.
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 people's formamidopyrimidine-DNA glycosylase 11.77 protein-actives, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
The invention provides a kind of polypeptide-human formamidopyrimidine-DNA glycosylase 11.77, it is basically by＜210〉aminoacid sequence shown in 2 forms.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 people's formamidopyrimidine-DNA glycosylase 11.77.As used herein, term " fragment ", " derivative " and " analogue " are meant biological function or the active polypeptide that keeps people's formamidopyrimidine-DNA glycosylase of the present invention 11.77 identical 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 by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.
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＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.
The polynucleotide of the mature polypeptide of coding＜210〉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 and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.
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 people formamidopyrimidine-DNA glycosylase 11.77.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding people formamidopyrimidine-DNA glycosylase 11.77 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., Molecular Cloning, A LaboratoryManual, 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 people formamidopyrimidine-DNA glycosylase 11.77; (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 people's formamidopyrimidine-DNA glycosylase 11.77 genetic expressions and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to 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 the host cell that produces through genetically engineered with the carrier of the present invention or formamidopyrimidine-DNA glycosylase 11.77 encoding sequences of directly choosing, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, the polynucleotide sequence of coding people formamidopyrimidine-DNA glycosylase 11.77 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 coding people formamidopyrimidine-DNA glycosylase 11.77 and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; 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, the polynucleotide of coding people formamidopyrimidine-DNA glycosylase 11.77 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 people's formamidopyrimidine-DNA glycosylase 11.77 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of everybody formamidopyrimidine-DNA glycosylase 11.77 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.
Formamidopyrimidine-DNA glycosylase is the enzyme that a kind of DNA of participation repairs, thereby its excision oxidation purine bases discharges 2,6-diamino-4-hydroxyl-5N-methyl formamido group pyrimidine (Fapy) and 7,8-dihydro-8-oxo guanine (8-OxoG) residue.FPG can also cut DNA in five purine pyrimidine sites (AP site) except having the glycosylase activity.The DNA repair ability of formamidopyrimidine-DNA glycosylase has guaranteed the genetic information fidelity of reproduction.
Discover that the repair mechanism of formamidopyrimidine-DNA glycosylase is identical with the uridylic glycosylase.Formamidopyrimidine-DNA glycosylase is very important for the existence of organism.If the gene of these enzymes is undergone mutation, people's physical appearance will be very poor, can catch diseases such as various cancerous swellings.
Polypeptide of the present invention is the polypeptide that contains the characteristic sequence of formamidopyrimidine-DNA glycosylase protein family, and its abnormal expression will cause the unusual of DNA repairing effect, and its AP site inscribe is active unusual, and produces relevant disease.This shows that the abnormal expression of formamidopyrimidine-DNA glycosylase 37 of the present invention will produce the especially various tumours of various diseases, fetal development disorders, dysplasia disease, immunological disease.
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) people formamidopyrimidine-DNA glycosylase 11.77.Agonist improves people's formamidopyrimidine-DNA glycosylase 11.77 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 expressing human formamidopyrimidine-DNA glycosylase 11.77 be cultivated with people's formamidopyrimidine-DNA glycosylase 11.77 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of people's formamidopyrimidine-DNA glycosylase 11.77 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of people's formamidopyrimidine-DNA glycosylase 11.77 can combine and eliminate its function with people's formamidopyrimidine-DNA glycosylase 11.77, or suppress the generation of this polypeptide, or combine 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, people's formamidopyrimidine-DNA glycosylase 11.77 can be added in the bioanalysis mensuration, interactional influence between people's formamidopyrimidine-DNA glycosylase 11.77 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 people's formamidopyrimidine-DNA glycosylase 11.77 bonded peptide molecules obtains.During screening, generally tackle people's formamidopyrimidine-DNA glycosylase 11.77 molecules 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 people's formamidopyrimidine-DNA glycosylase 11.77 antigenic determinants.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.
Can the choose method of formamidopyrimidine-DNA glycosylase 11.77 direct injection immune animals (as rabbit, mouse, rat etc.) of the production of polyclonal antibody 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 people formamidopyrimidine-DNA glycosylase 11.77 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-people's formamidopyrimidine-DNA glycosylase 11.77.
The antibody of anti-people's formamidopyrimidine-DNA glycosylase 11.77 can be used in the immunohistochemistry technology, detects the people's formamidopyrimidine-DNA glycosylase 11.77 in the biopsy specimen.
With the also available labelled with radioisotope of people's formamidopyrimidine-DNA glycosylase 11.77 bonded monoclonal antibodies, 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 people's formamidopyrimidine-DNA glycosylase 11.77 high-affinities 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 people's formamidopyrimidine-DNA glycosylase 11.77 positive cells.
The disease that antibody among the present invention can be used for treating or prevention and people's formamidopyrimidine-DNA glycosylase 11.77 are relevant.The antibody that gives suitable dosage can stimulate or block the generation or the activity of people's formamidopyrimidine-DNA glycosylase 11.77.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people formamidopyrimidine-DNA glycosylase 11.77 levels.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.People's formamidopyrimidine-DNA glycosylase 11.77 levels that detected in the test can be with laying down a definition the importance of people's formamidopyrimidine-DNA glycosylase 11.77 in various diseases and be used to the disease of diagnosing people's formamidopyrimidine-DNA glycosylase 11.77 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 people formamidopyrimidine-DNA glycosylase 11.77 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 people's formamidopyrimidine-DNA glycosylase 11.77 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for people's formamidopyrimidine-DNA glycosylase 11.77 of expressing variation, to suppress endogenic people's formamidopyrimidine-DNA glycosylase 11.77 activity.For example, a kind of people's formamidopyrimidine-DNA glycosylase 11.77 of variation can be the people's formamidopyrimidine-DNA glycosylase 11.77 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 11.77 expression of people's formamidopyrimidine-DNA glycosylase 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 people formamidopyrimidine-DNA glycosylase 11.77 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding people formamidopyrimidine-DNA glycosylase 11.77 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding people formamidopyrimidine-DNA glycosylase 11.77 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 people's formamidopyrimidine-DNA glycosylase 11.77mRNA 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 people formamidopyrimidine-DNA glycosylase 11.77 can be used for the diagnosis with the relative disease of people's formamidopyrimidine-DNA glycosylase 11.77.The unconventionality expression of the expression that the polynucleotide of coding people formamidopyrimidine-DNA glycosylase 11.77 can be used for detecting people's formamidopyrimidine-DNA glycosylase 11.77 people's formamidopyrimidine-DNA glycosylase 11.77 whether or under morbid state.As the dna sequence dna of the people's formamidopyrimidine-DNA glycosylase 11.77 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of people's formamidopyrimidine-DNA glycosylase 11.77.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.Personnel selection formamidopyrimidine-DNA glycosylase 11.77 special primers carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect people's formamidopyrimidine-DNA glycosylase 11.77.
The sudden change that detects people's formamidopyrimidine-DNA glycosylase 11.77 genes also can be used for diagnosing the relevant disease of people's formamidopyrimidine-DNA glycosylase 11.77.The form of people's formamidopyrimidine-DNA glycosylase 11.77 sudden change comprises that the point mutation compared with normal wild type people formamidopyrimidine-DNA glycosylase 11.77DNA sequence, 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 Manual ofBasic Techniques, Pergamon Press, New York (1988).
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
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.People's formamidopyrimidine-DNA glycosylase 11.77 comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to people's formamidopyrimidine-DNA glycosylase 11.77 of patient will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
Description of drawings
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 gene chip expression spectral comparison diagram of formamidopyrimidine-DNA glycosylase 11.77 of the present invention and formamidopyrimidine-DNA glycosylase.Last figure is the express spectra folding side figure of formamidopyrimidine-DNA glycosylase 11.77, and the below sequence is the express spectra folding side figure of formamidopyrimidine-DNA glycosylase.Wherein, 1-tire brain, 2-normal brain activity, 3-tire kidney, 4-liver cancer, 5-tire liver, 6-adult liver, 7-Tiroidina, 8-tire lung, 9-lung, 10-muscle, 11-thymus gland, 12-glioma.
Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating formamidopyrimidine-DNA glycosylase 11.77.11.77kDa be 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:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people's formamidopyrimidine-DNA glycosylase 11.77
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 5001F08 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 5001F08 clone is 918bp (as＜210〉1 shown in), from 287bp to 610bp the open reading frame (ORF) of a 324bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-5001F08, and the name of encoded protein matter is people's formamidopyrimidine-DNA glycosylase 11.77.
Embodiment 2: with the gene of RT-PCR method clones coding people formamidopyrimidine-DNA glycosylase 11.77
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’-GGGGAGCCTGTGGACGTGGCATGT-3’
Primer2：5’-AGCTTAAAAACCAAAAGGCAGAAA-3’
Primer1 for to be positioned at＜the forward sequence that begins of 1bp of 210〉15 ' end;
Primer2 be＜210〉1 in 3 ' end reverse sequence.
The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-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 the dna sequence dna and＜210 of PCR product〉1-918bp shown in 1 is identical.
Embodiment 3:Northern blotting analyst formamidopyrimidine-DNA glycosylase 11.77 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 people's formamidopyrimidine-DNA glycosylase 11.77 coding region sequences 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 4: the vivoexpression of recombinant human formamidopyrimidine-DNA glycosylase 11.77, separation and purifying
According to＜210〉1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:
Primer?3：5’-CCCCATATGATGGCAGCCTTTGGTGGGCCGCTG-3’
Primer?4：5’-CCCGAATTCCTAGTGTCCCCCCAGCGAGCTTGC-3’
5 ' end of these two sections primers contains NdeI and EcoRI restriction enzyme site respectively, is respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, and NdeI and EcoRI restriction enzyme site are corresponding to the expression vector plasmid
PET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-5001F08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-5001F08 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 NheI 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-5001F08) 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-5001F08) 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, obtained the target protein people formamidopyrimidine-DNA glycosylase 11.77 of purifying with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product).Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 11.77kDa 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 are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.
Embodiment 5 anti-people's formamidopyrimidine-DNA glycosylase 11.77 production of antibodies
Synthesize following people's formamidopyrimidine-DNA glycosylase 11.77 specific polypeptide with Peptide synthesizer (PE company product):
NH2-Met-Ala-Ala-Phe-Gly-Gly-Pro-Leu-Trp-Pro-Ala-Arg-Leu-Cys-Ser-COOH
With this polypeptide respectively with the coupling of hemocyanin and bovine serum albumin form compound, method 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's formamidopyrimidine-DNA glycosylase 11.77 specifically.
Embodiment 6DNA Microarray
Gene chip or gene micromatrix (DNA Microarray) are that present many National Laboratories and big drugmaker are all in the new technology of setting about developing and developing, it is meant and is arranged in a large amount of target fragments on the carriers such as glass, silicon in an orderly manner, to high-density, carry out the comparison and the analysis of data then with fluoroscopic examination and computer software, to reach purpose quick, efficient, that bioinformation is analyzed on high-throughput ground.Polynucleotide of the present invention can be used as target DNA and are used for biochip technology and are used for high-throughput and study new gene function; Seek and the new gene of the screening tissue specificity new gene of disease-related such as tumour particularly; The diagnosis of disease is as heredopathia.The existing in the literature multiple report of its concrete grammar step is as consulting document DeRisi, J.L., Lyer, V.﹠amp; Brown, P.O. (1997) Science278,680-686. and document Helle, R.A., Schema, M., Chai, A., Shalom, D., (1997) PNAS 94:2150-2155.
(1) point sample
Various full-length cDNA amounts to 4000 polynucleotide sequences as target DNA, comprising polynucleotide of the present invention.They are increased by PCR respectively, behind the purifying gained amplified production its concentration is transferred to about 500ng/ul, (available from U.S. Cartesian company) puts on glass medium with Cartesian 7500 point sample instruments, and distance between points is 280 μ m.Slide behind the point sample is carried out hydration, drying, places UV-crosslinked instrument crosslinked, and the wash-out after drying is fixed on DNA and is prepared into chip on the sheet glass.The existing in the literature multiple report of its concrete grammar step, the point sample post-processing step of present embodiment is:
1. hydration 4 hours in the wet environment;
2.0.2%SDS washed 1 minute;
3.ddH
2The O washed twice, each 1 minute;
4.NaBH
4Sealed 5 minutes;
5.95 in ℃ water 2 minutes;
6.0.2%SDS washed 1 minute;
7.ddH
2Twice of O flushing;
8. airing, 25 ℃ to be stored in the dark place standby.
(2) probe mark
With the single stage method total mRNA of extracting from human body mixed structure and body particular organization (or through stimulated cells strain) respectively, and with Oligotex mRNA Midi Kit (available from QiaGen company) purified mRNA, by reverse transcription respectively with fluorescent reagent Cy3dUTP (5-Amino-propargyl-2 '-deoxyuridine5 '-triphate coupledto Cy3 fluorescent dye, available from Amersham Phamacia Biotech company) mRNA of mark human body mixed structure, with fluorescent reagent Cy5dUTP (5-Amino-propargyl-2 '-deoxyuridine5 '-triphatecoupled to Cy5 fluorescent dye, available from Amersham Phamacia Biotech company) mRNA of mark body particular organization (or through stimulated cells strain), prepare probe after purified.Concrete steps reference and method are seen:
Schena，
M.，Shalon，D.，Heller，R.(1996)Proc.Natl.Acad.Sci.USA.Vol.93：10614-10619.Schena，M.，Shalon，Dari.，Davi?s，R.W.(1995)Science.270.(20)：467-480.
(3) hybridization
Respectively will be from the probe of above two kinds of tissues with chip at UniHyb
TMHybridized 16 hours in HybridizationSolution (available from the TeleChem company) hybridization solution, room temperature washings (1 * SSC, 0.2%SDS) the washing back is scanned with ScanArray 3000 scanners (available from U.S. General Scanning company), the image of scanning carries out data analysis with Imagene software (U.S. Biodiscovery company) to be handled, and calculates the Cy3/Cy5 ratio of each point.
Be respectively normal brain activity, liver cancer, muscle, tire brain, tire kidney, tire lung, tire liver, Tiroidina, thymus gland, adult liver, lung, glioma with upper machine body particular organization (or through stimulated cells strain).Draw folding side figure according to these 12 Cy3/Cy5 ratios.(Fig. 1).Formamidopyrimidine-DNA glycosylase 11.77 of the present invention as seen from the figure is very similar with the formamidopyrimidine-DNA glycosylase express spectra.
Sequence table
＜110〉Bode Gene Development Co., Ltd., Shanghai
The polynucleotide of＜120〉a kind of new polypeptide--people's formamidopyrimidine-DNA glycosylase 11.77 and this peptide species of coding
<130>5001F08
<160>2
<170>PatentIn?version?3.1
<210>1
<211>918
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(287)..(610)
<223>
<400>1
ggggagcctg?tggacgtggc?atgtggcgtg?gaccacatgg?tgaccctggc?caagtcattc 60
atctaaacct?ccctcacctg?cttgggcggc?cccgtcccgg?gaaccactgg?cactccttgg 120
cagaggctag?cgcgtggcca?gccccccggg?gttcttggat?ggtggtggcg?gaggaccctg 180
cgtgcagtgt?gacgctctgt?cctgaatccc?ttagcgggta?cctaccagga?ggatccgggc 240
aaggtccctc?tccagctgca?ggtgaggcct?gcggaactca?gcttgg?atg?gca?gcc 295
Met?Ala?Ala
1
ttt?ggt?ggg?ccg?ctg?tgg?ccc?gca?cgt?ctc?tgt?tct?ctc?caa?gta?aca 343
Phe?Gly?Gly?Pro?Leu?Trp?Pro?Ala?Arg?Leu?Cys?Ser?Leu?Gln?Val?Thr
5 10 15
tgc?gac?ggt?gtc?tgg?tgt?cac?gtc?tcg?cct?gag?aag?ccc?gtc?tta?gga 391
Cys?Asp?Gly?Val?Trp?Cys?His?Val?Ser?Pro?Glu?Lys?Pro?Val?Leu?Gly
20 25 30 35
aag?ctt?agc?ttg?aac?aca?gtg?ctc?ggg?agg?ttt?ctg?ctc?tgt?ctg?tca 439
Lys?Leu?Ser?Leu?Asn?Thr?Val?Leu?Gly?Arg?Phe?Leu?Leu?Cys?Leu?Ser
40 45 50
tgg?cag?tct?ctt?ggt?ttg?tgt?ctg?gcc?aag?gcc?atg?cgt?gtg?cct?cgg 487
Trp?Gln?Ser?Leu?Gly?Leu?Cys?Leu?Ala?Lys?Ala?Met?Arg?Val?Pro?Arg
55 60 65
acc?gag?ccc?cag?ctt?agg?cga?ggg?agt?cag?gct?ggc?ttc?ggc?cct?cgg 535
Thr?Glu?Pro?Gln?Leu?Arg?Arg?Gly?Ser?Gln?Ala?Gly?Phe?Gly?Pro?Arg
70 75 80
ttt?tca?ttc?agg?cca?ccc?tgc?cca?tgg?ccc?ttc?ctg?gcc?gcc?tgc?cac 583
Phe?Ser?Phe?Arg?Pro?Pro?Cys?Pro?Trp?Pro?Phe?Leu?Ala?Ala?Cys?His
85 90 95
acc?gca?agc?tcg?ctg?ggg?gga?cac?tag?aagcaccgtg?gcctgggatt 630
Thr?Ala?Ser?Ser?Leu?Gly?Gly?His
100 105
ccatctggag?ctgtccgcag?gcaccagccc?cagcctccca?ccacgctcac?tgcctggctt 690
ggaaaagtta?agaagcccct?caggaagaga?atcgaggcta?agttcctctg?cgccgagggc 750
cccgagcata?tccgccaagg?ctcagctgca?gtgccaggcg?gaggaggaag?atccagaaat 810
tgtgaacaat?gtttgattta?gtagcgtgac?ttgcctttcc?ctttaaaaac?atcttttaca 870
aatctgtctt?ggaataaagt?ctattttctg?ccttttggtt?tttaagct 918
<210>2
<211>107
<212>PRT
<213>Homo?sapiens
<400>2
Met?Ala?Ala?Phe?Gly?Gly?Pro?Leu?Trp?Pro?Ala?Arg?Leu?Cys?Ser?Leu
1 5 10 15
Gln?Val?Thr?Cys?Asp?Gly?Val?Trp?Cys?His?Val?Ser?Pro?Glu?Lys?Pro
20 25 30
Val?Leu?Gly?Lys?Leu?Ser?Leu?Asn?Thr?Val?Leu?Gly?Arg?Phe?Leu?Leu
35 40 45
Cys?Leu?Ser?Trp?Gln?Ser?Leu?Gly?Leu?Cys?Leu?Ala?Lys?Ala?Met?Arg
50 55 60
Val?Pro?Arg?Thr?Glu?Pro?Gln?Leu?Arg?Arg?Gly?Ser?Gln?Ala?Gly?Phe
65 70 75 80
Gly?Pro?Arg?Phe?Ser?Phe?Arg?Pro?Pro?Cys?Pro?Trp?Pro?Phe?Leu?Ala
85 90 95
Ala?Cys?His?Thr?Ala?Ser?Ser?Leu?Gly?Gly?His
100 105
Claims (10)
1, a kind of isolated polypeptide-people's formamidopyrimidine-DNA glycosylase 11.77 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉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＜210〉2.
3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉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＜210〉2 shown in the polynucleotide of the polypeptide of aminoacid sequence 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, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.
6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 287-610 position sequence or＜210〉1 in the sequence of 1-918 position.
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 people's formamidopyrimidine-DNA glycosylase 11.77 active polypeptide is characterized in that described method comprises:
(a) under expressing human formamidopyrimidine-DNA glycosylase 11.77 conditions, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have people's formamidopyrimidine-DNA glycosylase 11.77 active polypeptide.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with people's formamidopyrimidine-DNA glycosylase 11.77 specificity bonded antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031150772A CN1519320A (en) | 2003-01-21 | 2003-01-21 | Polypeptide-human formamido pyrimidine-glycosylase 11.77 and polynucleotide for encoding polypoptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031150772A CN1519320A (en) | 2003-01-21 | 2003-01-21 | Polypeptide-human formamido pyrimidine-glycosylase 11.77 and polynucleotide for encoding polypoptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1519320A true CN1519320A (en) | 2004-08-11 |
Family
ID=34284120
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031150772A Pending CN1519320A (en) | 2003-01-21 | 2003-01-21 | Polypeptide-human formamido pyrimidine-glycosylase 11.77 and polynucleotide for encoding polypoptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1519320A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025123662A1 (en) * | 2023-12-13 | 2025-06-19 | 广州达安基因股份有限公司 | Method for preparing formamidopyrimidine dna glycosylase |
-
2003
- 2003-01-21 CN CNA031150772A patent/CN1519320A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025123662A1 (en) * | 2023-12-13 | 2025-06-19 | 广州达安基因股份有限公司 | Method for preparing formamidopyrimidine dna glycosylase |
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