CN1493596A - Polypeptide-human programming cell death protein 22 and polynucleotide for coding said polypeptide - Google Patents
Polypeptide-human programming cell death protein 22 and polynucleotide for coding said polypeptide Download PDFInfo
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Abstract
A polypeptide-human programmed cell death protein 22, 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 antagonist of said polypeptide and its treating 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 polypeptide-human apoptosis 4 protein 22, 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
Apoptosis (PCD) is the elimination of multicellular organisms special cell type in growth course, is (1) of strict regulation and control under the physiological condition.Apoptosis is undertaken by apoptosis, and apoptosis refer to the cell that carries out apoptosis can observed metamorphosis.Comprise that cytolemma generation vesicle, cell shrinkage, chromosome condensation and DNA decompose (2).This process has reached the peak when cell debris becomes apoptotic body, in vivo the cytophagy (3) around the very fast quilt of these apoptotic bodies.Apoptosis is the disappearance mode of many unwanted cells of organism, to the ripe of fetal development, form generation, inflammation elimination, hematopoiesis and immunocyte and keep the cell number of healthy tissues and organ constant with the growth balance, so that the body aging aspect its important effect, so the form of complete, normal physiological function aspect apoptosis is kept to(for) multicellular organism play important effect (4) if the generation (5) that apoptosis makes a mistake and can cause tumour generation, autoimmune disease and AIDS.
Apoptosis be at suicide signal (suicide signals) by having triggered the signal conduction with combining of its acceptor, caused necrocytosis at last.Many molecules can be regulated dead path or the conduction of control dead signal.The nucleus that constitutes apoptosis mechanism is very conservative (6) from the nematode to people with relevant molecule mechanism.The cde3 of nematode, cde4, three genes and apoptosis-related of cde9.The Mammals aspect, people have focused on interleukin 1 β (IL-1 β) saccharase associated protein enzyme aspect to attention, this is a family that comprises 10 kinds of Mammals L-Cysteine HCL Anhydrouss at least, they are referred to as caspase, these proteolytic enzyme have caused the apoptosis of cell, show as: 1, these proteolytic enzyme of overexpression can cause apoptosis; 2, cell activates these proteolytic enzyme by classification and causes necrocytosis (7).
In addition: many evidences show that the change control apoptosis aspect of intracellular calcium concentration plays an important role.Dna fragmentationization is an apoptotic key character, and its formation relies on calcium ion activated endonuclease.Bringing out the apoptotic while also makes intracellular calcium ion concn raise, intracellular calcium ion chelator can suppress apoptosis by cell, under Fas stimulated, calcium ion discharged from intracellular establishment hiding-place rapidly, illustrates that there is the step (8) of calcium ion sensitivity in apoptotic process.
Pasquale, Vito (9) etc. have used the system of a dead trap ＂ of they called after ＂ in order to understand the gene relevant with apoptosis ".Its principle is; With the mouse cDNA library infected mice T-quadroma (3DO) that is structured in mammalian expression vector pLTP, should be able to protect some cells to avoid the apoptosis that external factor brings out.They have screened 6 cDNA clones as a result, and called after ALG-1 can suppress the apoptosis that TCR brings out to ALG-6 (Apoptosis-linked genes).Wherein ALG-2 is a kind of calcium binding protein, its existence and apoptosis-related.Transfection the T cell of Antisense cDNA (thereby suppressed ALG-2 albumen produce) can resist the apoptosis that many factors are brought out.
ALG-2 is made up of a polypeptide chain, and molecular weight is 22KDa, has comprised 5 calcium ion binding sites that are called the EF arm.In vitro study shows: under the existence condition, ALG-2 does not form weak special dimer to calcium ion.Having lacked carboxyl terminal or N-terminal ALG-2 can also show that ALG-2 has two structural domains in conjunction with calcium ion, and there are two calcium ion binding sites in N end structure territory, and the C end has three calcium ion binding sites, and the 5th EF arm is relevant with dimeric formation.Calcium ion can not be certain for the gathering of ALG-2, and calcium ion makes the N-end of ALG-2 and C-end conformation that variation (7) take place with combining of ALG-2.
Because ALG-2 is to form shape to express in mouse 3DO apoptosis process, so ALG-2 is by the mechanism that relies on calcium ion play a role in the downstream of caspase (9).In order to seek the interactional albumen with ALG-2, Missoto M utilizes research of co-immunoprecipitation method and ALG-2 interacting proteins.The result shows that ALG-2 is not having to form homodimer with self under the condition of calcium ion, ALG-2 and a kind of new protein-Alix (ALG-2 interacting protein X) form heterodimer under the condition that has calcium ion to exist, and after showing calcium ion and ALG-2 combining, their could interact (10).
In addition, the PROTEIN B RO1 of Alix and S.cerevisiae has very high homology (10), and BRO1 is a member of PKclp-Map kinase pathways, and BRO1 undergos mutation back and causes temperature sensitive sudden change, and this sudden change can be suppressed by calcium ion.Surfaces A LG-2/Alix may assault play a role (8) by activating the signal relevant with map kinase.
Because people's apoptosis 4 protein 22 albumen plays an important role in regulating body critical functions such as cell fission and fetal development as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify people's apoptosis 4 protein 22 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new person's apoptosis 4 protein 22 protein coding gene also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.
Summary of the invention
An object of the present invention is to provide isolating new polypeptide-human apoptosis 4 protein 22 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 apoptosis 4 protein 22.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain coding people apoptosis 4 protein 22.
Another object of the present invention provides the method for producing people's apoptosis 4 protein 22.
Another object of the present invention provides the antibody at polypeptide-human apoptosis 4 protein 22 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 invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:
(a) coding has＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences;
(b) with polynucleotide (a) complementary polynucleotide;
(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 97% homogeny.
More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have＜210〉1 in the sequence of 14-580 position; (b) have＜210〉1 in the sequence of 1-3758 position.
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.
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 apoptosis 4 protein 22, 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 apoptosis 4 protein 22.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps people's apoptosis 4 protein 22 of the present invention identical basically with " analogue ".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.The polynucleotide sequence total length that it comprises is 3758 bases, its open reading frame (14-580) 188 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the PROTEIN B RO1 of this polypeptide and S.cerevisiae has 96% homology, deducibility goes out the similar 26S Proteasome Structure and Function of PROTEIN B RO1 that this people's apoptosis 4 protein 22 has S.cerevisiae.
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 apoptosis 4 protein 22.
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 apoptosis 4 protein 22 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 people apoptosis 4 protein 22; (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 apoptosis 4 protein 22 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 the host cell that produces through genetically engineered with the carrier of the present invention or the apoptosis 4 protein 22 encoding sequence 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 apoptosis 4 protein 22 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 apoptosis 4 protein 22 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 people apoptosis 4 protein 22 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 apoptosis 4 protein 22 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of everybody apoptosis 4 protein 22 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 above-mentioned disease that these are caused owing to the cell transition apoptosis, can utilize blocking-up apoptotic signal transduction path and realizing, thereby suppress the formation of Alix or use to combine the inhibition apoptosis at the inhibitor blocking-up Alix of Alix and ALG-2 as sense-rna with Alix.
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) people apoptosis 4 protein 22.Agonist improves people's apoptosis 4 protein 22 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 apoptosis 4 protein 22 be cultivated with people's apoptosis 4 protein 22 of mark.Measure the medicine raising then or check this interactional ability.
The antagonist of people's apoptosis 4 protein 22 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of people's apoptosis 4 protein 22 can combine and eliminate its function with people's apoptosis 4 protein 22, 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, people's apoptosis 4 protein 22 can be added in the bioanalysis mensuration, interactional influence between people's apoptosis 4 protein 22 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 apoptosis 4 protein 22 bonded peptide molecule obtains.During screening, generally tackle people's apoptosis 4 protein 22 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 people's apoptosis 4 protein 22 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.
Can the choose method of apoptosis 4 protein 22 direct injection immune animal (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 apoptosis 4 protein 22 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 apoptosis 4 protein 22.
The antibody of anti-people's apoptosis 4 protein 22 can be used in the immunohistochemistry technology, detects the people's apoptosis 4 protein 22 in the biopsy specimen.
With the also available labelled with radioisotope of people's apoptosis 4 protein 22 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 people's apoptosis 4 protein 22 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 people's apoptosis 4 protein 22 positive cells.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people's apoptosis 4 protein 22.The antibody that gives suitable dosage can stimulate or block the generation or the activity of people's apoptosis 4 protein 22.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people apoptosis 4 protein 22 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people's apoptosis 4 protein 22 level that is detected in the test can be with laying down a definition the importance of people's apoptosis 4 protein 22 in various diseases and be used to the disease of diagnosing people's apoptosis 4 protein 22 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 apoptosis 4 protein 22 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 apoptosis 4 protein 22 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for people's apoptosis 4 protein 22 of expressing variation, to suppress endogenic people's apoptosis 4 protein 22 activity.For example, a kind of people's apoptosis 4 protein 22 of variation can be the people's apoptosis 4 protein 22 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 expression of people's apoptosis 4 protein 22 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 apoptosis 4 protein 22 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding people apoptosis 4 protein 22 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding people apoptosis 4 protein 22 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 apoptosis 4 protein 22 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 people apoptosis 4 protein 22 can be used for the diagnosis with the relative disease of people's apoptosis 4 protein 22.The unconventionality expression of the expression that the polynucleotide of coding people apoptosis 4 protein 22 can be used for detecting people's apoptosis 4 protein 22 people's apoptosis 4 protein 22 whether or under morbid state.As the dna sequence dna of people's apoptosis 4 protein 22 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of people's apoptosis 4 protein 22.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.The special primer of personnel selection apoptosis 4 protein 22 carries out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect people's apoptosis 4 protein 22.
The sudden change that detects people's apoptosis 4 protein 22 gene also can be used for the disease of diagnosing people's apoptosis 4 protein 22 relevant.The form of people's apoptosis 4 protein 22 sudden change comprises that the point mutation compared with normal wild type people apoptosis 4 protein 22 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, Mendelian Inheritancein 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.People's apoptosis 4 protein 22 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 apoptosis 4 protein 22 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 amino acid sequence homology comparison diagram of the PROTEIN B RO1 of inventor's apoptosis 4 protein 22 and S.cerevisiae.The top sequence is people's apoptosis 4 protein 22, and the below sequence is the PROTEIN B RO1 of S.cerevisiae.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 people's apoptosis 4 protein 22.22kDa 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:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people's apoptosis 4 protein 22
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 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 0453A12 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 0453A12 clone is 3758bp (as＜210〉1 shown in), from 14bp to 580bp the open reading frame (ORF) of a 567bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-0453A12, and the name of encoded protein matter is people's apoptosis 4 protein 22.
Embodiment 2:cDNA clone's homology retrieval
With the sequence and the encoded protein sequence thereof of people's apoptosis 4 protein 22 of the present invention, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with people's apoptosis 4 protein 22 homology of the present invention is the PROTEIN B RO1 of a kind of known S.cerevisiae, and its encoded protein number is AJ005074 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 96%; Similarity is 98%.
Embodiment 3: with the gene of RT-PCR method clones coding people apoptosis 4 protein 22
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’CTATGTTTCATGTATGAAGAAAAG-3’(<210>3)
Primer2：5’-AATCTTTCAACTTTTATTTCTAG-3’(<210>4)
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〉1-3758bp shown in 1 is identical.
Embodiment 4:Northern blotting analyst apoptosis 4 protein 22 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 apoptosis 4 protein 22 coding region sequence (14bp to 580bp) 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 apoptosis 4 protein 22
According to＜210〉1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:
Primer3：5’-CCCGGATCCATGAAGAAAAGAAAAGCTGATATT-3’(<210>5)
Primer4：5’-CCCCTCGAGTTACTGCTGTGGATAGTAAGACTGC-3’(<210>6)
5 ' end of these two sections primers contains BamHI and XhoI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, BamHI and XhoI 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-0453A12 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0453A12 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 BamHI and Xhol, 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-0453A12) 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-0453A12) 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 people apoptosis 4 protein 22 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 22kDa 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 6 anti-people's apoptosis 4 protein 22 production of antibodies
With the synthetic specific polypeptide of following people's apoptosis 4 protein 22: the MH of Peptide synthesizer (PE company product)
2-Met-Lys-Lys-Arg-Lys-Ala-Asp-Ile-Leu-Cys-Val-Leu-Ile-Phe-Ser-COOH.
Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, etal.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 apoptosis 4 protein 22 specifically.
Sequence table
＜110〉Bode Gene Development Co., Ltd., Shanghai
The polynucleotide of＜a 120〉peptide species--people's apoptosis 4 protein 22 and this peptide species of coding
<130>0453a12
<160>6
<170>Patentln?version3.1
<210>1
<211>660
<212>DNA
<213>Homo?sapiens
<220>
<221>CDS
<222>(14)..(580)
<223>
<400>1
ctatgtttca?tgt?atg?aag?aaa?aga?aaa?gct?gat?att?tta?tgt?gtg?tta 49
Met?Lys?Lys?Arg?Lys?Ala?Asp?Ile?Leu?Cys?Val?Leu
1 5 10
att?ttt?tct?atc?atg?ttg?att?ttt?aac?act?ctg?tct?ttg?ata?agg?gac 97
Ile?Phe?Ser?Ile?Met?Leu?Ile?Phe?Asn?Thr?Leu?Ser?Leu?Ile?Arg?Asp
15 20 25
ttg?caa?caa?agc?att?gcc?aga?gaa?cct?agt?gct?cct?tca?att?cct?aca 145
Leu?Gln?Gln?Ser?Ile?Ala?Arg?Glu?Pro?Ser?Ala?Pro?Ser?Ile?Pro?Thr
30 35 40
cct?gcg?tat?cag?tcc?tta?cca?gca?gga?gga?cat?gca?cca?act?cct?cca 193
Pro?Ala?Tyr?Gln?Ser?Leu?Pro?Ala?Gly?Gly?His?Ala?Pro?Thr?Pro?Pro
45 50 55 60
act?cca?gcg?cca?aga?acc?atg?ccg?cct?act?aag?ccc?cag?ccc?cca?gcc 241
Thr?Pro?Ala?Pro?Arg?Thr?Met?Pro?Pro?Thr?Lys?Pro?Gln?Pro?Pro?Ala
65 70 75
agg?cct?cca?cca?cct?gtg?ctt?cca?gca?aat?cga?gct?cct?tct?gct?act 289
Arg?Pro?Pro?Pro?Pro?Val?Leu?Pro?Ala?Asn?Arg?Ala?Pro?Ser?Ala?Thr
80 85 90
gct?cca?tct?cca?gtg?ggg?gct?ggg?act?gct?gcg?cca?gct?cca?tca?caa 337
Ala?Pro?Ser?Pro?Val?Gly?Ala?Gly?Thr?Ala?Ala?Pro?Ala?Pro?Ser?Gln
95 100 105
acg?cct?ggc?tca?gct?cct?cct?cca?cag?gcg?cag?gga?cca?ccc?tat?ccc 385
Thr?Pro?Gly?Ser?Ala?Pro?Pro?Pro?Gln?Ala?Gln?Gly?Pro?Pro?Tyr?Pro
110 115 120
acc?tat?cca?gga?tat?cct?ggg?tat?tgc?caa?atg?ccc?atg?ccc?atg?ggc 433
Thr?Tyr?Pro?Gly?Tyr?Pro?Gly?Tyr?Cys?Gln?Met?Pro?Met?Pro?Met?Gly
125 130 135 140
tat?aat?cct?tat?gcg?tat?ggc?cag?tat?aat?atg?cca?tat?cca?cca?gtg 481
Tyr?Asn?Pro?Tyr?Ala?Tyr?Gly?Gln?Tyr?Asn?Met?Pro?Tyr?Pro?Pro?Val
145 150 155
tat?cac?cag?agt?cct?gga?cag?gct?ccg?tac?ccg?gga?ccc?cag?cag?cct 529
Tyr?His?Gln?Ser?Pro?Gly?Gln?Ala?Pro?Tyr?Pro?Gly?Pro?Gln?Gln?Pro
160 165 170
tca?tac?ccc?ttc?cct?cag?ccc?cca?cag?cag?tct?tac?tat?cca?cag?cag 577
Ser?Tyr?Pro?Phe?Pro?Gln?Pro?Pro?Gln?Gln?Ser?Tyr?Tyr?Pro?Gln?Gln
175 180 185
taa?tatgtctgct?cagcagctca?gctgattcag?atcagaggga?aagaaatacc 630
aaccctgcaa taagtgtact aagctctacg
660
<210>2
<211>188
<212>PRT
<213>Homo?sapiens
<400>2
Met?Lys?Lys?Arg?Lys?Ala?Asp?Ile?Leu?Cys?Val?Leu?Ile?Phe?Ser?Ile
1 5 10 15
Met?Leu?Ile?Phe?Asn?Thr?Leu?Ser?Leu?Ile?Arg?Asp?Leu?Gln?Gln?Ser
20 25 30
Ile?Ala?Arg?Glu?Pro?Ser?Ala?Pro?Ser?Ile?Pro?Thr?Pro?Ala?Tyr?Gln
35 40 45
Ser?Leu?Pro?Ala?Gly?Gly?His?Ala?Pro?Thr?Pro?Pro?Thr?Pro?Ala?Pro
50 55 60
Arg?Thr?Met?Pro?Pro?Thr?Lys?Pro?Gln?Pro?Pro?Ala?Arg?Pro?Pro?Pro
65 70 75 80
Pro?Val?Leu?Pro?Ala?Asn?Arg?Ala?Pro?Ser?Ala?Thr?Ala?Pro?Ser?Pro
85 90 95
Val?Gly?Ala?Gly?Thr?Ala?Ala?Pro?Ala?Pro?Ser?Gln?Thr?Pro?Gly?Ser
100 105 110
Ala?Pro?Pro?Pro?Gln?Ala?Gln?Gly?Pro?Pro?Tyr?Pro?Thr?Tyr?Pro?Gly
115 120 125
Tyr?Pro?Gly?Tyr?Cys?Gln?Met?Pro?Met?Pro?Met?Gly?Tyr?Asn?Pro?Tyr
130 135 140
Ala?Tyr?Gly?Gln?Tyr?Asn?Met?Pro?Tyr?Pro?Pro?Val?Tyr?His?Gln?Ser
145 150 155 160
Pro?Gly?Gln?Ala?Pro?Tyr?Pro?Gly?Pro?Gln?Gln?Pro?Ser?Tyr?Pro?Phe
165 170 175
Pro?Gln?Pro?Pro?Gln?Gln?Ser?Tyr?Tyr?Pro?Gln?Gln
180 185
<210>3
<211>24
<212>DNA
<213>Homo?sapiens
<400>3
ctatgtttca tgtatgaaga aaag
24
<210>4
<211>23
<212>DNA
<213>Homo?sapiens
<400>4
aatctttcaa cttttatttc tag
23
<210>5
<211>33
<212>DNA
<213>Homo?sapiens
<400>5
cccggatcca tgaagaaaag aaaagctgat att
33
<210>6
<211>34
<212>DNA
<213>Homo?sapiens
<400>6
cccctcgagt tactgctgtg gatagtaaga ctgc
34
Claims (10)
1, a kind of isolated polypeptide-people's apoptosis 4 protein 22 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 97% 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 14-580 position sequence or＜210〉1 in the sequence of 1-3758 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 the active polypeptide of people's apoptosis 4 protein 22 is characterized in that described method comprises:
(a) under expressing human apoptosis 4 protein 22 condition, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have the active polypeptide of people's apoptosis 4 protein 22.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with people's apoptosis 4 protein 22 specificity bonded antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021377332A CN1493596A (en) | 2002-10-30 | 2002-10-30 | Polypeptide-human programming cell death protein 22 and polynucleotide for coding said polypeptide |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021377332A CN1493596A (en) | 2002-10-30 | 2002-10-30 | Polypeptide-human programming cell death protein 22 and polynucleotide for coding said polypeptide |
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| Publication Number | Publication Date |
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|---|---|---|---|
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| CN (1) | CN1493596A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353377B (en) * | 2008-09-06 | 2011-08-31 | 苏州大学 | Zone 1 protein of programmed cell death protein 2 analogue and uses thereof |
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2002
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353377B (en) * | 2008-09-06 | 2011-08-31 | 苏州大学 | Zone 1 protein of programmed cell death protein 2 analogue and uses thereof |
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