CN1297897A - New human oxyglutelinoid and its code sequence - Google Patents

Abstract

The present invention discloses a novel polypeptide, hGRX-Bio36, polynucleotides encoding this polypeptide and DNA recombination process to produce the polypeptide. The present invention also discloses the method of applying the polypeptide in treating various diseases, such as genital system disease, cardiovascular system disease, cerebral ischemia and cerebral nerve cell damage and immunological diseases. The present invention also discloses the antagonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this polypeptide.

Description

New people's oxyglutelinoid and encoding sequence thereof

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide one people's oxyglutelinoid (human glutaredoxin-Bio36 abbreviates " hGRX-Bio36 " as), 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.

(glutaredoxin GRX) is called sulfurtransferase (thioltransferase) again to the paddy oxidation protein, is one of member of redox protein extended familys, and it is a kind of protein of small molecular weight, extensively is present in prokaryotic organism and the most eukaryotes.All contain following one section conservative bioactive sequence in the gene of coding paddy oxidation protein (GRX): " Cys-Pro-Try-Cys-".

Pass through electrophoretic mobility shift assay, known road wild-type paddy oxidation protein (GRX that obtains after the non-reorganization), can be by the oxidized dna binding activity of repairing a kind of transcription factor PEBP2 of diamide, and jointly the dna binding activity of PEBP2 is carried out redox modulating (Nakamura T with sulphur oxidation egg, et al, Free Radic Res1999 Oct; 31 (4): 357-65).Find that by the intravital paddy oxidation protein of mouse (GRX) being carried out expression study paddy oxidation protein (GRX) also has the critical function that cell is taken precautions against oxidative stress.The gene of coding paddy oxidation protein GRX can be expressed in cerebral neuron cell, endotheliocyte, tanycyte and the ependymocyte of mouse and be obtained GRXmRNA and paddy oxidation protein (GRX), wherein in the taper neurocyte in hippocampus CA3 zone, express the highest, when the obstruction of artery of midbrain, the expression of GRX mRNA promptly descends.By paddy oxidation protein (GRX) is carried out immunohistochemical analysis, find Interhemispheric local asphyxia and nerve cell damage (the Takagi Y that all can cause the expression of GRX promptly to descend, et al, Biochem Biophys Res Commun 1999 May 10; 258 (2): 390-4).

In addition, paddy oxidation protein (GRX) is being played the part of important role (Garcia-Pardo L, et al.Mol Hum Reprod 1999Oct as a kind of cell antioxidant in the lutein cell ovulation working cycle of human body ovary; 5 (10): 914-919).

Therefore, with the treatment research and development paddy oxidation protein (GRX) polypeptide of purpose and antibody thereof, stimulant, inhibitor, on physiology, pharmacology, significance is arranged.

The oxyglutelinoid (abbreviating " hGRX-Bio36 " as) that an object of the present invention is to provide isolating new polypeptide-human 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 of the oxyglutelinoid (hGRX-Bio36) that contains the people that encodes.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide of the oxyglutelinoid (hGRX-Bio36) that contains the people that encodes.

Another object of the present invention provides the method for the oxyglutelinoid (hGRX-Bio36) of producing the people.

Another object of the present invention provides the antibody of the oxyglutelinoid (hGRX-Bio36) at polypeptide-human of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor of the oxyglutelinoid (hGRX-Bio36) at polypeptide-human of the present invention.

Another object of the present invention provides the method for the diagnosis disease relevant unusually with treatment and people's oxyglutelinoid (hGRX-Bio36).

In a first aspect of the present invention, the isolated people's of novelty oxyglutelinoid (hGRX-Bio36) is provided, this polypeptide is the people source, and it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of these polypeptide of separated coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people's of coding oxyglutelinoid (hGRX-Bio36); (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 16-999 position among the SEQ ID NO:1; (b) has the sequence of 1-2265 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

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

As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating people's oxyglutelinoid (hGRX-Bio36) " is meant that people's oxyglutelinoid (hGRX-Bio36) is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying people's of standard oxyglutelinoid (hGRX-Bio36).Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of people's oxyglutelinoid (hGRX-Bio36) polypeptide can be used amino acid sequence analysis.

The invention provides a kind of oxyglutelinoid (hGRX-Bio36) of new polypeptide-human, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of people's oxyglutelinoid (hGRX-Bio36).As used herein, term " fragment ", " derivative " are meant identical biological function or the active polypeptide of oxyglutelinoid (hGRX-Bio36) that keeps people of the present invention basically with " analogue ".The fragment of polypeptide of the present invention, derivative or analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue) (i) are arranged, and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong within the known scope of those skilled in the art.

The present invention also provides the nucleic acid (polynucleotide) of separated coding hGRX-Bio36 of the present invention.In an example, this nucleic acid is made up of the polynucleotide that coding has a polypeptide of SEQ ID NO:2 aminoacid sequence substantially.Preferably, polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1, and these polynucleotide are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 2265 bases, 327 amino acid whose hGRX-Bio36 of its open reading frame (16-999 position) coding.Relatively find according to amino acid sequence homologous, Z46787 on this polypeptide and the nematode C.Elegans karyomit(e) III has 51% homology, and the cDNA sequence of this polypeptide of encoding contains the conservative base of GRX gene family, and deducibility goes out this new people paddy oxidation protein (hGRX-Bio36) and has the similar 26S Proteasome Structure and Function of GRX family.The cDNA sequence of hGRX-Bio36 of the present invention also contains C3HC4 type zinc finger protein (ring finger) gene conservative base simultaneously, therefore may contain the partial function of zinc finger protein.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ IDNO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is a kind of 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) when hybridization add denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be at least 30 Nucleotide, be more preferably at least 50 Nucleotide, 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's oxyglutelinoid (hGRX-Bio36).

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's of the present invention oxyglutelinoid (hGRX-Bio36) 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 polymerase chain reaction technique, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of mensuration people's oxyglutelinoid (hGRX-Bio36) transcript; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.Dna probe can carry out mark with radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of people's oxyglutelinoid (hGRX-Bio36) genetic expression 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 or the nucleotide sequence of its various dna fragmentations, and available 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.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 oxyglutelinoid (hGRX-Bio36) encoding sequence of carrier of the present invention or directly personnel selection, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence of coding people's oxyglutelinoid (hGRX-Bio36) 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 of the oxyglutelinoid (hGRX-Bio36) that contains the people that encodes and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.MolecularCloning, 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's oxyglutelinoid (hGRX-Bio36) 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.Another kind method is to use MgCl ₂Handle.If 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.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), utilize polynucleotide sequence of the present invention to can be used to express or produce the people's of reorganization oxyglutelinoid (hGRX-Bio36).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people's oxyglutelinoid (hGRX-Bio36), or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat reproductive system disease, cardiovascular disorder, neural cell injury and the caused disease of immune system disorder etc.

Paddy oxidation protein hGRX-Bio36GRX is preferable over the treatment brain nervous cell especially anti-oxidation stress and the metabolism disorder of adjustment of treatment corpus luteum is caused disease.

The present invention also provides SCREENED COMPOUND to identify the method for medicament that improves (agonist) or check (antagonist) people's oxyglutelinoid (hGRX-Bio36).The oxyglutelinoid (hGRX-Bio36) that agonist improves people 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, mammalian cell be cultivated with the albumen of the present invention of mark.Measure the medicine raising then or check the proteic ability of the present invention.

The antagonist of people's oxyglutelinoid (hGRX-Bio36) comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of people's oxyglutelinoid (hGRX-Bio36) can combine and eliminate its function with people's oxyglutelinoid (hGRX-Bio36) polypeptide, 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 oxyglutelinoid (hGRX-Bio36) can be added during bioanalysis measures, interactional influence between people's oxyglutelinoid (hGRX-Bio36) 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 oxyglutelinoid (hGRX-Bio36) bonded peptide molecule obtains.During screening, oxyglutelinoid (hGRX-Bio36) molecule of generally tackling the people carries 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 of oxyglutelinoid (hGRX-Bio36) antigenic determinant at the people.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The method of the oxyglutelinoid that the production of polyclonal antibody can be chosen (hGRX-Bio36) direct injection immune animal (as rabbit, mouse, rat etc.) obtains.There is multiple adjuvant to 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's oxyglutelinoid (hGRX-Bio36) 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 oxyglutelinoid (hGRX-Bio36).

The antibody of anti-people's oxyglutelinoid (hGRX-Bio36) can be used in the immunohistochemistry technology, detects the oxyglutelinoid (hGRX-Bio36) of the people in the biopsy specimen.

With people's the also available labelled with radioisotope of oxyglutelinoid (hGRX-Bio36) 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 oxyglutelinoid (hGRX-Bio36) 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 oxyglutelinoid (hGRX-Bio36) positive cells.

Antibody among the present invention can be used for treating or prevention and people's the relevant disease of oxyglutelinoid (hGRX-Bio36).The antibody that gives suitable dosage can stimulate or block the generation or the activity of people's oxyglutelinoid (hGRX-Bio36).

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people's oxyglutelinoid (hGRX-Bio36) level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people's who is detected in the test oxyglutelinoid (hGRX-Bio36) level can be with the people's that lays down a definition the importance of oxyglutelinoid (hGRX-Bio36) in various diseases and the disease that is used to diagnose people's oxyglutelinoid (hGRX-Bio36) 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's oxyglutelinoid (hGRX-Bio36) 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 oxyglutelinoid (hGRX-Bio36) or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the people's who expresses variation oxyglutelinoid (hGRX-Bio36), with oxyglutelinoid (hGRX-Bio36) activity that suppresses endogenic people.For example, a kind of people's of variation oxyglutelinoid (hGRX-Bio36) can be the oxyglutelinoid (hGRX-Bio36) that shortens, lacked the people in 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 people's oxyglutelinoid (hGRX-Bio36) expression or the disease of active caused by abnormal.Deriving from the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding people's oxyglutelinoid (hGRX-Bio36) are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding people's paddy oxidation protein (hGRX-Bio36) be found in existing document (Sambrook, etal.).The polynucleotide of reorganization coding people's oxyglutelinoid (hGRX-Bio36) can be packaged in the liposome and then 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 people oxyglutelinoid hGRX-Bio36 mRNA oligonucleotide (comprising sense-rna and DNA) and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Coding people's the polynucleotide of oxyglutelinoid (hGRX-Bio36) can be used for diagnosing the relevant disease of oxyglutelinoid (hGRX-Bio36) with the people.The unconventionality expression of the expression that coding people's the polynucleotide of oxyglutelinoid (hGRX-Bio36) can be used for detecting people paddy oxidation protein (hGRX-Bio36) oxyglutelinoid (hGRX-Bio36) of people whether or under morbid state.As the dna sequence dna of the people's that encodes oxyglutelinoid (hGRX-Bio36) can be used for biopsy specimen is hybridized expression situation with the oxyglutelinoid (hGRX-Bio36) of judging the people.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 oxyglutelinoid (hGRX-Bio36) of personnel selection carries out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect people's oxyglutelinoid (hGRX-Bio36).

The sudden change that detects people's oxyglutelinoid (hGRX-Bio36) gene also can be used for diagnosing people's the relevant disease of oxyglutelinoid (hGRX-Bio36).The form of people's oxyglutelinoid (hGRX-Bio36) sudden change comprises that the point mutation compared with normal wild type people's oxyglutelinoid (hGRX-Bio36) 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).Yet, have only chromosomal marker thing seldom to can be used for the marker chromosomes position now 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 oxyglutelinoid (hGRX-Bio36) comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's people's oxyglutelinoid (hGRX-Bio36) will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

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

Fig. 1 is the inventor's oxyglutelinoid (hGRX-Bio36) and the amino acid sequence homology comparison diagram of the Z46787 on the nematode C.elegans karyomit(e).The top sequence is a people's of the present invention oxyglutelinoid (hGRX-Bio36), and the below sequence is Z46787.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 oxyglutelinoid (hGRX-Bio36).36kDa 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 oxyglutelinoid (hGRX-Bio36)

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 Quick mRNA separating 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 the sequence of all clones' 5 ' and 3 ' end 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 (Genbank) measured are compared, found that the cDNA sequence of one of them clone 0945c09 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 0945c09 clone is 2265bp (shown in SEQ ID NO:1), from 16bp to 999bp the open reading frame (ORF) of a 983bp, the new protein (shown in SEQ ID NO:2) of encoding arranged.This clone is named as pBS-0945c09, and the name of encoded protein matter is people's oxyglutelinoid (hGRX-Bio36).

Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of people's of the present invention oxyglutelinoid (hGRX-Bio36), 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.With people's of the present invention oxyglutelinoid (hGRX-Bio36) gene that homology is the highest be a fragment gene on a kind of known nematode karyomit(e) III, its encoded protein number is Z46787 in the access of Genbank, this nematode protein is similar to the paddy oxidation protein, and has C3H4 type zinc and refer to.Both protein homologies the results are shown in Fig. 1, both height homologies, and its homogeny is 51%; Similarity is 69%.

Embodiment 3: the gene of using RT-PCR method clones coding people's oxyglutelinoid (hGRX-Bio36)

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:

Primer 1:5 '-GGGCGAGCCG TGAAGATGGC-3 ' (SEQ ID NO:3)

Primer 2: 5 '-CCATCTTCAC GGCTCGCCC-3 ' (SEQ ID NO:4)

The forward sequence that primer 1 begins corresponding to the 5 ' lbp that holds that is positioned at SEQ ID NO:1;

Primer 2 is corresponding to 3 ' the end reverse sequence of SEQ ID NO:1.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of beta-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-2265bp shown in the SEQ ID NO:1 are identical.

Embodiment 4:Northern blotting analyst's oxyglutelinoid (hGRX-Bio36) 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 the people's of pcr amplification shown in Figure 1 oxyglutelinoid (hGRX-Bio36) coding region sequence (16bp to 999bp).Will ³²The probe of p-mark (about 2 * 10 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.

Example 5: vivoexpression, separation and the purifying of the oxyglutelinoid of recombinant human (hGRX-Bio36)

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

Primer 3:5-GGGCCCATGGCGGCAGTGGTGGAG-3 (SEQ ID No:5)

Primer 4:5-CATATGCCAGGATGTAGTTGATGCC-3 (SEQ ID No:6)

5 ' end of these two sections primers contains ApaI and NdeI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, ApaI and NdeI 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-0945c09 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0945c09 plasmid 10pg, primer-3 and primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantagepolymerase 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 NcoI 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-0945c09) 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-0945c09) 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 target protein one people's of purifying oxyglutelinoid (hGRX-Bio36) with 6 Histidines (6His-Tag) bonded affinity column His.Bind QuickCartridge (Novagen company product).Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 36kDa place.

This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.

Embodiment 6 anti-people's oxyglutelinoid (hGRX-Bio36) production of antibodies

The specific polypeptide of oxyglutelinoid (hGRX-Bio36) with the synthetic following people of Peptide synthesizer (PE company product):

NH ₂-Cys?Pro?Tyr?Cys?Lys?Glu?Lys?Val?Asp?Leu-COOH(SEQ?ID?NO：7)。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 oxyglutelinoid (hGRX-Bio36) specifically.

Embodiment 7: the antisense oligonucleotide of people's oxyglutelinoid (hGRX-Bio36) suppresses the propagation of HL-60 cell

Adopt 392 type the dna synthesizers antisense oligonucleotide (AS-ODN) and the positive MODN (S-ODN) of synthetic people's oxyglutelinoid (hGRX-Bio36) automatically, and with tetraethylthiuram disulfide (Tetraethylthiuram disulfide, TETD) carry out chemically modified and become AS-PS-ODN and S-PS-ODN, its base sequence is:

AS-PS-ODN：5-CTCCACCACTGCCGCCAT-3(SEQ?ID?NO.8)

S-PS-ODN：?5-ATGGCGGCAGTGGTGGAG-3(SEQ?ID?NO.9)

With concentration is 5x10 ⁵The HL-60 cell be divided into 3 groups at 37 ℃, 5%CO ₂Cultivate in the incubator: (1) control group is simple HL-60 cell; (2) HL-60 cell+AS-PS-ODN; (3) HL-60 cell+S-PS-ODN.The final concentration of AS-PS-ODN or S-PS-ODN is 20 μ g/ml.Cell cultures detects the mortality ratio of cell with tetrabromophenol sulfonphthalein dyeing after 24 hours.The result shows that it is 91.8% that AS-PS-ODN is organized cell mortality, and control group and S-PS-ODN group mortality ratio are 4.5%.Show that the antisense oligonucleotide of people's oxyglutelinoid (hGRX-Bio36) gene can suppress the propagation of HL-60 cell effectively.

Sequence table

(1) general information:

(i) applicant:

(A) name: Shengyuan Gene Development Co., Ltd., Shanghai

(B) street: No. 668 610 Room, East Road, Beijing

(C) city: Shanghai

(E) country: China

(F) postcode: 200001

(ii) denomination of invention: new people's oxyglutelinoid and encoding sequence thereof

(iii) sequence number: 9

(2) information of SEQ ID NO:1:

(i) sequence signature:

(A) length: 2265bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO：1：GGGCGAGCCG TGAAGATGGC GGCAGTGGTG GAGGTGGAGG TTGGAGGTGG TGCTGCTGGG 60GAACGGGAGC TGGATGAGGT TGATATGTCA GATCTCTCTC CAGAAGAGCA ATGGAGGGTC 120GAGCACGCAC GCATGCATGC CAAGCACCGT GGCCATGAAG CTATGCATGC TGAAATGGTC 180CTCATCCTCA TCGCAACCTT GGTGGTGGCC CAGCTGCTCC TGGTGCAGTG GAAGCAGAGG 240CACCCACGCT CCTACAATAT GGTGACCCTC TTTCAGATGT GGGTTGTTCC CCTCTATTTC 300ACAGTGAAGC TGCACTGGTG GAGGTTCCTA GTGATCTGGA TCTTGTTCTC TGCTGTCACA 360GCCTTTGTTA CCTTCCGAGC CACCCGAAAA CCTCTAGTAC AGACAACCCC AAGGTTGGTT 420TATAAGTGGT TCCTGCTAAT CTATAAAATC AGCTATGCCA CTGGCATTGT TGGCTACATG 480GCTGTCATGT TTACCCTCTT TGGTCTTAAC TTATTATTCA AGATCAAACC AGAAGATGCC 540ATGGACTTTG GCATCTCCCT TCTCTTCTAT GGCCTCTACT ATGGAGTTCT GGAACGGGAC 600TTTGCAGAAA TGTGTGCAGA CTACATGGCA TCTACCATAG GGTTCTACAG CGAGTCGGGC 660ATGCCTACCA AACATCTTTC AGACAGTGTG TGTGCTGTGT GTGGGCAGCA GATCTTTGTG 720GACGTCAGTG AAGAGGGGAT CATTGAGAAC ACGTATAGGC TGTCCTGCAA TCATGTCTTC 780CACGAGTTCT GCATCCGTGG CTGGTGCATC GTGGGAAAGA AGCAAACGTG TCCCTACTGC 840AAAGAGAAGG TAGACCTCAA GAGGATGTTC AGCAATCCCT GGGAGAGGCC TCACGTCATG 900TATGGGCAAC TGCTGGACTG GCTTCGATAC TTGGTAGCCT GGCAGCCTGT CATCATTGGT 960GTAGTCCAAG GCATCAACTA CATCCTGGGC CTGGAATAGT GATGAAGAGC ATCAGTGGAA 1020AACCCACCCC ACACGCCATG GACCTCAGGG CACTCTCCTC CCTGCCCACA AAGACCTCCT 1080GGGTGGGAAA GACTCAAAGG GGCGCTTGGG CCACTCAGGA CCCCTCCGGC TGTGTCGGAC 1140TGGGGAGGGA TATGATGGAG AGCCAGCCAG TGGGGCTGTC AGCAGTGGGG GGCTTTTTAA 1200AAGAAAACTA TTTTGATGAA TATATTTAAA AAACCTTTTT TTATTGTGGA GCATAGGAAT 1260TGCCCCCCTC CAGGCTTCAC CCTCCCTGCC TAAGCAGGGT GGGGGCAGAG CCATGACATT 1320TTTGGTTTAA AGGAGCCTTC TCATCTCTGG CCGAGAACAC TGCTGGGCTC CCAGGTAGCT 1380GAAGGCCTCA GCCCACCCAC TCCCTTCTTC CCTGTGTGGG GCTCAAGCCT GGTGCACTTA 1440GTATAGAAGA GCTAACGTAC TCAGGCTTGT GCCACGGGTG AGCACTGAGG CCCCAGGTGT 1500CCTCCCTCCC CCACTGTCTG GGACACAGGA CAGGTGTTGC GTCTGACTCT GTCTCCCCCT 1560GTCCAAGAGC TATAGGCTGG TTGAGGCAAG TGGAACCTTA TCAGTGACTT GGCCAACGAG 1620GGAAGCCTTG GGGCCTCAGA ATTCATTGAA AATTTGTTTT GGGACAACTG GAAGAATCAG 1680CACCATGGAT ACTCACCCGT GGGCAGGGCT GTGGTCAGCC AAGAATAGAG GTCATATAGT 1740TGGGCCATGG GGCAATTGTT GGCAGCCATG ACTGACGGCC CCTTGTGCAA GCTCATTCTT 1800CAGTGGATCC TTCAGTGGAG TGGTGACACG TGCCAGCGGT CACTCTGCCA CATCACTTCC 1860TTCTTGAAAG CCTAAGAGTG CGTATGCGTG TGTGTGTGTG TGTGTGTACG TGAGGCAGAA 1920GAATATGACA GATGTCAGCT TCTCCCTGTG TCAGATGATA TGTCCTTGAA GCTGGCAGGG 1980GATTCCCAGT CCAGGGAATA TCCCTACATG ATTTTTGTCA TTCATGGGTC CTCCCTGGGC 2040CTGACAACGG GAGTGGTGGG TGGGTGAGAA CAGGTTCTGA GAGGGGGCTT CTAGGAAGGC 2100CCCAGCCCTA GTCCTAGGCG ACTTGAATAA AGTGGGAGGT CAAACATGGG CCTTCGGCAT 2160TGGGTTTCTA GGGAATATGG CTCAGTAGGG CCTGCCCCTT CAAGGGCAGC AAGAACGAAG 2220CAGGATGTTG TCACTCCCGT CTGAATAAAG GGCGAGCCGT GAAGATGG 2265

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 327 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO：2：Met Ala Ala Val Val Glu Val Glu Val Gly Gly Gly Ala Ala Gly 15Glu Arg Glu Leu Asp Glu Val Asp Met Ser Asp Leu Ser Pro Glu 30Glu Gln Trp Arg Val Glu His Ala Arg Met His Ala Lys His Arg 45Gly His Glu Ala Met His Ala Glu Met Val Leu Ile Leu Ile Ala 60Thr Leu Val Val Ala Gln Leu Leu Leu Val Gln Trp Lys Gln Arg 75His Pro Arg Ser Tyr Asn Met Val Thr Leu Phe Gln Met Trp Val 90Val Pro Leu Tyr Phe Thr Val Lys Leu His Trp Trp Arg Phe Leu 105Val Ile Trp Ile Leu Phe Ser Ala Val Thr Ala Phe Val Thr Phe 120Arg Ala Thr Arg Lys Pro Leu Val Gln Thr Thr Pro Arg Leu Val 135Tyr Lys Trp Phe Leu Leu Ile Tyr Lys Ile Ser Tyr Ala Thr Gly 150Ile Val Gly Tyr Met Ala Val Met Phe Thr Leu Phe Gly Leu Asn 165Leu Leu Phe Lys Ile Lys Pro Glu Asp Ala Met Asp Phe Gly Ile 180Ser Leu Leu Phe Tyr Gly Leu Tyr Tyr Gly Val Leu Glu Arg Asp 195Phe Ala Glu Met Cys Ala Asp Tyr Met Ala Ser Thr Ile Gly Phe 210Tyr Ser Glu Ser Gly Met Pro Thr Lys His Leu Ser Asp Ser Val 225Cys Ala Val Cys Gly Gln Gln Ile Phe Val Asp Val Ser Glu Glu 240Gly Ile Ile Glu Asn Thr Tyr Arg Leu Ser Cys Asn His Val Phe 255His Glu Phe Cys Ile Arg Gly Trp Cys Ile Val Gly Lys Lys Gln 270Thr Cys Pro Tyr Cys Lys Glu Lys Val Asp Leu Lys Arg Met Phe 285Ser Asn Pro Trp Glu Arg Pro His Val Met Tyr Gly Gln Leu Leu 300Asp Trp Leu Arg Tyr Leu Val Ala Trp Gln Pro Val Ile Ile Gly 315Val Val Gln Gly Ile Asn Tyr Ile Leu Gly Leu Glu 327 ( 2 ) SEQ ID NO：3 ( i )

(A) length: 20 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:3:GGGCGAGCCG TGAAGATGGC 20

(2) information of SEQ ID NO:4

(i) sequence signature

(A) length: 19 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:4:CCATCTTCAC GGCTCGCCC 19

(2) information of SEQ ID NO:5

(i) sequence signature

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:5:GGGCCCATGG CGGCAGTGGT GGAG 24

(2) information of SEQ ID NO:6

(i) sequence signature

(A) length: base

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:6:CATATGCCAG GATGTAGTTG ATGCC 25

(2) information of SEQ ID NO:7:

(i) sequence signature:

(A) length: 10 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:7:Cys Pro Tyr Cys Lys Glu Lys Val Asp Leu 10

(2) information of SEQ ID NO:8

(i) sequence signature

(A) length: 18 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:8:

CTCCACCACT?GCCGCCAT 18

(2) information of SEQ ID NO:9

(i) sequence signature

(A) length: 18 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: oligonucleotide

(xi) sequence description: SEQ ID NO:9:

ATGGCGGCAG?TGGTGGAG 18

Claims (18)

1, a kind of isolating people's oxyglutelinoid hGRX-Bio36 is characterized in that it comprises: have the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

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

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

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

(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;

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

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

5, polynucleotide as claimed in claim 4 is characterized in that, described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQ ID NO:2.

6, polynucleotide as claimed in claim 4 is characterized in that, the sequence of described polynucleotide includes the sequence of 1-2265 position among the sequence of 16-999 position among the SEQ ID NO:1 or the SEQ ID NO:1.

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

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

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

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

9, a kind of preparation method with active polypeptide of oxyglutelinoid hGRX-Bio36 of people is characterized in that described method comprises:

(a) under the oxyglutelinoid hGRX-Bio36 of expressing human condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate the active polypeptide of oxyglutelinoid hGRX-Bio36 with people.

10, a kind of can with polypeptide bonded antibody, it is characterized in that, described antibody be can with people's oxyglutelinoid hGRX-Bio36 specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that, they are simulation, promotion, antagonism or the active compound that suppresses people's oxyglutelinoid hGRX-Bio36.

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

13, the described application of compound of a kind of claim 11 is characterized in that, the oxyglutelinoid hGRX-Bio36 that described compound is used for the mediator in vivo, the method for external activity.

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

As the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that 15, it is applied to screen stand-in, the agonist of people's oxyglutelinoid hGRX-Bio36, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

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

17, as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11, it is characterized in that, form pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as the relevant unusually disease of diagnosis or treatment and people's oxyglutelinoid hGRX-Bio36 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that, be used for the treatment of the medicine of reproductive system disease, cancer, cardiovascular disorder, cerebral ischemia and Cranial nerve injury as birth trauma and disease that immune system disorder causes with described polypeptide, polynucleotide or compound.

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