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CN1303931A - Novel polypeptide-XRN-100 and polynucleotide coding said polypeptide - Google Patents

Novel polypeptide-XRN-100 and polynucleotide coding said polypeptide Download PDF

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CN1303931A
CN1303931A CN 99119819 CN99119819A CN1303931A CN 1303931 A CN1303931 A CN 1303931A CN 99119819 CN99119819 CN 99119819 CN 99119819 A CN99119819 A CN 99119819A CN 1303931 A CN1303931 A CN 1303931A
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polypeptide
polynucleotide
xrn
sequence
seq
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毛裕民
谢毅
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BORONG GENE DEVELOPMENT CO LTD SHANGHAI
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BORONG GENE DEVELOPMENT CO LTD SHANGHAI
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Abstract

The present invention discloses a novel polypeptide-homologous recombination medium-chain exchange protein and exoribonuclease protein XRN-100, polynucleotide coding said polypeptide and its method for producing this polypeptide by using DNA recombination technology. Said invention also discloses the method for curing several diseases, such as chromosomal disorder due to genetic informatin change and error of germ-cell and somatic cell, chromosome breakage syndrome, various developmental disorder, some tumors, inflammation, immunological disease and blood disease, etc. by using said polypeptide. Said invention also discloses an antagonist resisting said polypeptide and its therapeutic effect. It also discloses the application of polynucleotide for coding this novel protein XRN-100.

Description

The polynucleotide of a kind of new polypeptide---XRN-100 and this peptide species of coding
The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---XRN-100, 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.
The XRN gene coded protein is the chain exchanger in the homologous recombination, has the exoribonuclease function simultaneously.
The biological site mutation of gene and the variation methods such as genetic recombination between the gene of relying on adapts to natural selection.Homologous recombination occurs between the homologous sequence of DNA.The dye exchange of daughter of the non-sisters of eukaryote, the dye exchange of daughter of sisters, the conversion of bacterium and some lower eukaryotes, the reorganization of phage all belongs to this type of.In homologous recombination, the interim homologous sequence pairing of presynapsis forms heterodimer DNA and causes chain exchange and gene recombination.The albumen of XRN genes encoding belongs to the proteinoid in the cell, RecA albumen in this proteinoid such as the intestinal bacteria (Escherichia coli), form DNA-albumen filament (filament) and seek the homology zone with the dna molecular of strand, make the homologous sequence pairing, form the heterodimer point of connection, by this proteic chain exchange interaction, the guiding homologous recombination.In the mitotic division of cell and the division of slowing down, brought into play important effect.
The XRN gene coded protein has the exoribonuclease activity simultaneously, plays a role in the metabolism of RNA.This proteins encoded tool 5 ＇ 3 ＇ hydrolysis 5 ＇ are the ability of the RNA of phosphate group.This fermentoid has following function: 1) in the mRNA metabolic turnover, hydrolysis 5 ＇ end is not with the mRNA of cap structure.2) relate to the shearing of ribosome-RNA(rRNA) 5 ＇ end and be processed to form sophisticated ribosome-RNA(rRNA).3) the downstream RNA product behind the hydrolysis polyA tailing site in the transcription pausing of mRNA.In the growing of cell, the XRN gene coded protein has been brought into play vital role.
An object of the present invention is to provide isolating new polypeptide---XRN-100 with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of this polypeptide of coding.
Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the XRN-100 that encodes.
Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the XRN-100 that encodes.
Another object of the present invention provides the method for producing XRN-100.
Another object of the present invention provides at polypeptide of the present invention---the antibody of XRN-100.
Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of XRN-100, antagonist, agonist, inhibitor.
Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with XRN-100.
In a first aspect of the present invention, novel isolated XRN-100 is provided, this polypeptide is the people source, it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 94% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned XRN-100 of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 60-2783 position among the SEQ ID NO:1; (b) has the sequence of 1-3271 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 XRN-100 " is meant that XRN-100 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 XRN-100 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of XRN-100 polypeptide can be used amino acid sequence analysis.
The invention provides a kind of new polypeptide---XRN-100, 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 XRN-100.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of XRN-100 of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.
The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 3271 bases, its open reading frame (60--2783) 907 amino acid of having encoded.Find relatively that according to amino acid sequence homologous the amino acid sequence homology of this polypeptide and mouse homologous protein Dhml reaches 93%, deducibility goes out this new XRN-100 and has the similar 26S Proteasome Structure and Function of Dhml.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as O.2 * SSC, 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, the length of " nucleic acid fragment " contain 10 Nucleotide at least, better are 20-30 Nucleotide at least, are 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 XRN-100.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
The special polynucleotide sequence of coding XRN-100 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 cDlNA 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 XRN-100; (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 XRN-100 genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.
Use method (Sailki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly with the host cell of XRN-100 encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.
Among the present invention, the polynucleotide sequence of coding XRN-100 can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.
Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna that contains the XRN-100 that encodes and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance etc.
Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.
Among the present invention, the polynucleotide of coding XRN-100 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 XRN-100 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people XRN-100, 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.
Because the XRN gene coded protein is the chain exchanger in the homologous recombination, has the exoribonuclease function simultaneously.So can infer that homologous recombination medium chain exchanger of the present invention and exoribonuclease albumin X RN-100 play an important role in the reduction division of sexual cell.The genetic information that can cause sexual cell unusually of this polypeptide gene and expression product thereof changes and even mistake, causes chromosomal disorder.These diseases include but not limited to: Klinefelter syndrome, XYY syndrome, XX male syndrome, XXX women's syndrome, Turner syndrome, mongolism, cat's cry syndrome, 13-patau syndrome, E trisomy, Fragile X syndrome, chromosome breakage syndrome, and other chromosome structure and numerical abnormality symptom.
Because the XRN gene coded protein is the chain exchanger in the homologous recombination, has the exoribonuclease function simultaneously, plays a role in the metabolism of RNA.So can infer that this polypeptide gene and expression product XRN-100 thereof also play an important role in somatic mitotic division.Disorders, some tumour, inflammation, immunological disease, blood disease, chromosome breakage syndrome are grown in can causing unusually of this polypeptide gene and expression product thereof.These diseases include but not limited to: spina bifida, the cranium fissure, anencephalia, the brain bulging, the hole deformity of brain, congenital hydrocephalus, the aqueduct deformity, achondroplastic dwarf's disease, spondyloepiphyseal dysplasia disease, the Langer-Giedion syndromes, hypogenitalism, epispadia, latent, with malformation syndrome of short and small stature such as Conradi syndromes and Danbolt-Closs syndromes, congenital glaucoma or cataract, congenital little fissura palpebrae, retinal development is unusual, atrophia nervi optici congenita, ulcuscuris, monster, the Williams syndromes, the Alagille syndromes, shellfish syndrome Weis two, the substrate epithelial tumor, squama shape epithelial tumor, mucinous tumors, fiber, lipoma, chondroma, vascular tumor, lymphoma, the hemopoietic tissue tumour, neuroma, adenoma, atopic reaction, adult respiratory distress syndrome, the lung eosinophilia, rheumatoid arthritis, similar rheumatism sample sacroiliitis, osteoarthritis, cholecystitis, glomerulonephritis, dermatomyositis, polymyositis, Addison's disease, louis-Bar syndrome, Bloom syndrome, the tint permanence xeroderma.
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 that sexual cell and somatic genetic information change so that mistake due to chromosomal disorder, chromosome breakage syndrome, various growth disorders, some tumour, inflammation, immunological disease, blood disease etc.
The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) XRN-100.Agonist improves XRN-100 biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the XRN-100 with mark cultivates with mammalian cell or the film preparation of expressing XRN-100.Measure the medicine raising then or check this interactional ability.
The antagonist of XRN-100 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of XRN-100 can combine and eliminate its function with XRN-100, 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, XRN-100 can be added during bioanalysis measures, by measuring compound interactional influence between XRN-100 and its acceptor is determined whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with XRN-100 bonded peptide molecule obtains.During screening, generally tackle the XRN-100 molecule and carry out mark.
The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody at the XRN-100 antigenic determinant.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.
The method of the available XRN-100 direct injection of the production of polyclonal antibody immune animal (as rabbit, mouse, rat etc.) obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of monoclonal antibody of preparation XRN-100 include but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the single-chain antibody of anti-XRN-100.
The antibody of anti-XRN-100 can be used in the immunohistochemistry technology, detects the XRN-100 in the biopsy specimen.
With the also available labelled with radioisotope of XRN-100 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 XRN-100 high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the XRN-100 positive cells.
The disease that antibody among the present invention can be used for treating or prevention is relevant with XRN-100.The antibody that gives suitable dosage can stimulate or block generation or the activity of XRN-100.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization XRN-100 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The XRN-100 level that is detected in the test can be with laying down a definition the importance of XRN-100 in various diseases and be used to the disease of diagnosing XRN-100 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 XRN-100 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 XRN-100 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the XRN-100 that expresses variation, to suppress endogenic XRN-100 activity.For example, a kind of XRN-100 of variation can be the XRN-100 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 XRN-100 expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding XRN-100 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding XRN-100 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding XRN-100 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 XRN-100mRNA 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 XRN-100 can be used for the diagnosis with the relative disease of XRN-100.The unconventionality expression of the expression that the polynucleotide of coding XRN-100 can be used for detecting XRN-100 XRN-100 whether or under morbid state.As the dna sequence dna of the XRN-100 that encodes can be used for biopsy specimen is hybridized to judge the expression situation of XRN-100.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect XRN-100 with the special primer of XRN-100.
The sudden change that detects the XRN-100 gene also can be used for the disease of diagnosing XRN-100 relevant.The form of XRN-100 sudden change comprises that the point mutation compared with normal wild type XRN-100 dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.
In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).
The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.
Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.
The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.
Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.XRN-100 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 XRN-100 will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the amino acid sequence homology comparison diagram of XRN-100 of the present invention and Dhml.The top sequence is XRN-100, and the below sequence is Dhml.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 XRN-100.99.8kDa be proteinic molecular weight.The arrow indication is isolated protein band.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.The clone of embodiment 1:XRN-100
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 Isoolation 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 ＇ ends 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 0049E09 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 0049E09 clone is 3271bp (shown in Seq ID NO:1), from 60bp to 2783bp the open reading frame (ORF) of a 907bp, the new protein (shown in SeqID NO:2) of encoding arranged.We are with this clone's called after pBS-0049E09, encoded protein matter called after XRN-100.
Embodiment 2:cDNA clone's homology retrieval
With sequence and the encoded protein sequence thereof of XRN-100 of the present invention, with Blast program (BasiclocalAlignment 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 XRN-100 homology of the present invention is a kind of known Dhml, and its encoded protein number is D38517 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 93%.Embodiment 3: with the gene of RT-PCR method clones coding XRN-100
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:
Primerl:?5’-GCCGTCTCTTTGGTTACGCTCG-3’(SEQ?ID?NO:3)
Primer2:?5’-TTTTAAAAGTTTAACTTTATTT-3’(SEQ?ID?NO:4)
Primerl is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;
Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.
The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl 2, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-3271bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting Analysis of X RN-100 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 XRN-100 coding region sequence (60bp to 2783bp) 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 PhosphorImager.Embodiment 5: vivoexpression, separation and the purifying of reorganization XRN-100
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’-CCCCATATGATGGGAGTCCCGGCGTTCTTCCG-3’(Seq?ID?No:5)
Primer?4:?5’-CCCCATATGATGGGAGTCCCGGCGTTCTTCCG-3’(Seq?ID?No:6)
5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to the selectivity restriction enzyme site on the expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.6986 5.3).With the pBS-0049E09 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0049E09 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 Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0049E09) 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-0049E09) 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 XRN-100 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 99.8kDa 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-XRN-100 production of antibodies
Synthesize the specific polypeptide of following XRN-100 with Peptide synthesizer (PE company product):
NH 2-Met-Gly-Val-Pro-Ala-Phe-Phe-Arg-Trp-Leu-Ser-Arg-Lys-Tyr-Pro-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemi stry, 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 XRN-100 specifically.
Sequence table (1) general information: (ⅱ) denomination of invention: new XRN-100 and encoding sequence thereof (ⅲ) sequence number: the information of 7 (2) SEQ ID NO:1: (ⅰ) sequence signature:
(A) length: 3271bp
(B) type: nucleic acid
(C) chain: two strands
( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:1: 1 GCCGTCTCTTTGGTTACGCTCGTCAGCCGGTCGGCCGCCGCCTCCAGCCGTGTGCCGCTA 61 TGGGAGTCCCGGCGTTCTTCCGCTGGCTCAGCCGCAAGTACCCGTCCATCATAGTCAACT121 GCGTGGAAGAGAAGACCAGCACCAAAAATGAAGATGAAATGATGGTTGCAATTTTTGAGT181 ACATTGACAGACTTTTCAGTATTGTAAGACCAAGAAGACTTCTCTACATGGCAATAGATG241 GAGTGGCACCACGTGCTAAAATGAACCAGCAGCGTTCAAGGAGGTTCAGGGCATCAAAAG301 AAGGAATGGAAGCAGCAGTCGAGAAGCAGCGAGTCAGGGAAGAAATATTGGCAAAAGGTG361 GCTTTCTTCCTCCAGAAGAAATAAAAGAAAGATTTGACAGCAACTGTATTACACCAGGAA421 CTGAATTCATGGACAATCTTGCTAAATGCCTTCGCTATTACATAGCTGATCGTTTAAATA481 ATGACCCTGGGTGGAAAAATTTGACAGTTATTTTATCTGATGCTAGTGCTCCTGGTGAAG541 GAGAACATAAAATCATGGATTACATTAGAAGGCAAAGAGCCCAGCCTAACCATGACCCAA601 ATACTCATCATTGTTTATGTGGAGCAGATGCTGATCTCATTATGCTTGGCCTTGCCACAC661 ATGAACCGAACTTTACCATTATTAGAGAAGAATTCAAACCAAACAAGCCCAAACCATGTG721 GTCTTTGTAATCAGTTTGGACATGAGGTCAAAGATTGTGAAGGTTTGCCAAGAGAAAAGA781 AGGGAAAGCATGATGAACTTGCCGATAGTCTTCCTTGTGCAGAAGGAGAGTTTATCTTCC841 TTCGGCTTAATGTTCTTCGTGAGTATTTGGAAAGAGAACTCACAATGGCCAGCCTACCAT901 TCACATTTGATGTTGAGAGGAGCATTGATGACTGGGTTTTCATGTGCTTCTTTGTGGGAA 961 ATGACTTCCTCCCTCATTTGCCATCGTTAGAGATTAGGGAAAATGCAATTGACCGTTTGG1021 TTAACATATACAAAAATGTGGTACACAAAACTGGGGGTTACCTTACAGAAAGTGGTTATG1081 TCAATCTGCAAAGAGTACAGATGATCATGTTAGCAGTTGGTGAAGTTGAGGATAGCATTT1141 TTAAAAAGAGAAAGGATGATGAGGACAGTTTTAGAAGACGACAGAAAGAAAAAAGAAAGA1201 GAATGAAGAGAGATCAACCAGCTTTCACTCCTAGTGGAATATTAACTCCTCATGCCTTGG1261 GTTCAAGAAATTCACCAGGTTCTCAAGTAGCCAGTAATCCGAGACAAGCAGCCTATGAAA1321 TGAGGATGCAGAATAACTCTAGTCCTTCGATATCTCCTAATACGAGTTTCACATCTGATG1381 GCTCCCCGTCTCCATTAGGAGGAATTAAGCGAAAAGCAGAAGACAGTGACAGTGAACCTG1441 AGCCAGAGGATAATGTCAGGTTATGGGAAGCTGGCTGGAAGCAGCGGTACTACAAGAACA1501 AATTTGATGTGGATGCAGCTGATGAGAAATTCCGTCGGAAAGTTGTGCAGTCGTACGTTG1561 AAGGACTTTGCTGGGTTCTTAGATATTATTACCAGGGCTGTGCTTCCTGGAAGTGGTATT1621 ATCCATTTCATTATGCACCATTTGCTTCAGACTTTGAAGGCATTGCAGACATGCCATCTG1681 ATTTTGAGAAGGGTACGAAACCGTTTAAACCACTAGAACAACTTATGGGGGTATTTCCAG1741 CTGCAAGTGGTAATTTTCTACCTCCATCATGGCGGAAGCTCATGAGTGATCCTGATTCTA1801 GTATAATTGACTTCTATCCTGAAGATTTTGCTATTGATTTGAATGGGAAGAAATATGCAT1861 GGCAAGGTGTTGCTCTCTTGCCATTCGTGGATGAGCGAAGGCTACGAGCTGCCCTAGAAG1921 AGGTATACCCAGACCTCACTCCAGAAGAGACCAGAAGAAACAGCCTTGGAGGTGATGTCT1981 TATTTGTGGGGAAACATCACCCACTCCATGACTTCATTTTAGAGCTGTACCAGACAGGTT2041 CCACAGAGCCAGTGGAGGTACCCCCTGAACTATGTCATGGGATTCAAGGAAAGTTTTCTT2101 TGGATGAAGAAGCCATTCTTCCAGATCAAATAGTATGTTCTCCTGTTCCTATGTTAAGGG2161 ATCTGACACAGAACACTGTAGTCAGTATTAATTTTAAAGACCCACAGCTTGCTGAAGATT2221 ACATTTTTAAAGCTGTAATGCTTCCAGGAGCAAGAAAGCCAGCAGCAGTACTGAAACCTA2281 GTGACTGGGAAAAATCCAGCAATGGACGGCAGTGGAAGCCTCAGCTTGGCTTTAACCGTG2341 ACCGGAGGCCTGTGCACCTGGATCAGGCAGCCTTCAGGACTTTGGGCCATGTGATGCCAA2401 GAGGCTCAGGAACTGGCATTTACAGCAATGCTGCACCACCACCTGTGACTTACCAGGGAA2461 ACTTATACAGGCCGCTTTTGAGAGGACAAGCCCAGATTCCAAAACTTATGTCAAATATGA2521 GGCCCCAGGATTCCTGGCGAGGTCCTCCTCTCCTTTTCCAGCAGCAAAGGTTTGACAGAG2581 GCGTTGGGGCTGAACCTCTGCTCCCATGGAACCGGATGCTGCAAACCCAGAATGCAGCCT2641 TCCAGCCAAACCAGTACCAGATGCTAGCTGGGCCTGGTGGGTATCCACCCAGACGAGATG2701 ATCGTGGAGGGAGACAGGGATATCCCAGAGAAGGAAGGAAATACCCTTTGCCACCACCCT2761 CAGGAAGATACAATTGGAATTAAGCTTTTGTAAAGCTTTCCCAAATCCTTTCATCATTCT2821 ACAGTTTTATGCTATTTGTGGAAAGATTTCTTTCTCAAGTAGTAGTTTTTAATAAAACTA2881 CAGTACTTTGTGTATTTCTTTTAACTGTGTATATTTCTACTGATCTGATCTCACTGTTTA2941 TGTTGCTTTCCAAAGATGTATGTTGCATAATACAGTGGATCTGAATTTATTATTGCTTAT3001 AAAACACATTTGATGGAATAGGAGTACTGGTTTTTCATAATGGTTAAAAATGAAACCAGC3061 TGTGGATTTCAAAACACAGTGTATTCTAGATCATCTAAGATCCATGCTGATTTTTATTGC3121 ACAAGAATTAGGTTTGAACTCTTGAGCTGGAACCTCAGCAAACTAGAGTATATATTGTTC3181 AGTATTTCTTTGGAAACATTTCATTAATGTACTTGTCTTACAGAAATTTCTGAACTTTAG3241 TAAAAAAAAAAATAAAGTTAAACTTTTAAAA ( 3 ) SEQ ID NO:2： ( ⅰ ) ：
(A) length: 907 amino acid
(B) type: amino acid
( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO:2: 1 Met Gly Val Pro Ala Phe Phe ArgTrp Leu Ser Arg Lys Tyr Pro 16 Ser Ile Ile Val Asn Cys Val Glu Glu Lys Thr Ser Thr Lys Asn 31 Glu Asp Glu Met Met Val Ala Ile Phe Glu Tyr Ile Asp Arg Leu 46 Phe Ser Ile Val Arg Pro Arg Arg Leu Leu Tyr Met Ala Ile Asp 61 Gly Val Ala Pro Arg Ala Lys Met Asn Gln Gln Arg Ser Arg Arg 76 Phe Arg Ala Ser Lys Glu Gly Met Glu Ala Ala Val Glu Lys Gln 91 Arg Val Arg Glu Glu Ile Leu Ala Lys Gly Gly Phe Leu Pro Pro106 Glu Glu Ile Lys Glu Arg Phe Asp Ser Asn Cys Ile Thr Pro Gly121 Thr Glu Phe Met Asp Asn Leu Ala Lys Cys Leu Arg Tyr Tyr Ile136 Ala Asp Arg Leu Asn Asn Asp Pro Gly Trp Lys Asn Leu Thr Val151 Ile Leu Ser Asp Ala Ser Ala Pro Gly Glu Gly Glu His Lys Ile166 Met Asp Tyr Ile Arg Arg Gln Arg Ala Gln Pro Asn His Asp Pro181 Asn Thr His His Cys Leu Cys Gly Ala Asp Ala Asp Leu Ile Met196 Leu Gly Leu Ala Thr His Glu Pro Asn Phe Thr Ile Ile Arg Glu211 Glu Phe Lys Pro Asn Lys Pro Lys Pro Cys Gly Leu Cys Asn Gln226 Phe Gly His Glu Val Lys Asp Cys Glu Gly Leu Pro Arg Glu Lys241 Lys Gly Lys His Asp Glu Leu Ala Asp Ser Leu Pro Cys Ala Glu256 Gly Glu Phe Ile Phe Leu Arg Leu Asn Val Leu Arg Glu Tyr Leu271 Glu Arg Glu Leu Thr Met Ala Ser Leu Pro Phe Thr Phe Asp Val286 Glu Arg Ser Ile Asp Asp Trp Val Phe Met Cys Phe Phe Val Gly301 Asn Asp Phe Leu Pro His Leu Pro Ser Leu Glu Ile Arg Glu Asn316 Ala lle Asp Arg Leu Val Asn Ile Tyr Lys Asn Val Val His Lys331 Thr Gly Gly Tyr Leu Thr Glu Ser Gly Tyr Val Asn Leu Gln Arg346 Val Gln Met Ile Met Leu Ala Val Gly Glu Val Glu Asp Ser Ile361 Phe Lys Lys Arg Lys Asp Asp Glu Asp Ser Phe Arg Arg Arg Gln376 Lys Glu Lys Arg Lys Arg Met Lys Arg Asp Gln Pro Ala Phe Thr391 Pro Ser Gly Ile Leu Thr Pro His Ala Leu Gly Ser Arg Asn Ser406 Pro Gly Ser Gln Val Ala Ser Asn Pro Arg Gln Ala Ala Tyr Glu421 Met Arg Met Gln Asn Asn Ser Ser Pro Ser Ile Ser Pro Asn Thr436 Ser Phe Thr Ser Asp Gly Ser Pro Ser Pro Leu Gly Gly Ile Lys451 Arg Lys Ala Glu Asp Ser Asp Ser Glu Pro Glu Pro Glu Asp Asn466 Val Arg Leu Trp Glu Ala Gly Trp Lys Gln Arg Tyr Tyr Lys Asn481 Lys Phe Asp Val Asp Ala Ala Asp Glu Lys Phe Arg Arg Lys Val496 Val Gln Ser Tyr Val Glu Gly Leu Cys Trp Val Leu Arg Tyr Tyr511 Tyr Gln Gly Cys Ala Ser Trp Lys Trp Tyr Tyr Pro Phe His Tyr526 Ala Pro Phe Ala Ser Asp Phe Glu Gly Ile Ala Asp Met Pro Ser541 Asp Phe Glu Lys Gly Thr Lys Pro Phe Lys Pro Leu Glu Gln Leu556 Met Gly Val Phe Pro Ala Ala Ser Gly Asn Phe Leu Pro Pro Ser571 Trp Arg Lys Leu Met Ser Asp Pro Asp Ser Ser lle Ile Asp Phe586 Tyr Pro Glu Asp Phe Ala Ile Asp Leu Asn Gly Lys Lys Tyr Ala601 Trp Gln Gly Val Ala Leu Leu Pro Phe Val Asp Glu Arg Arg Leu616 Arg Ala Ala Leu Glu Glu Val Tyr Pro Asp Leu Thr Pro Glu Glu631 Thr Arg Arg Asn Ser Leu Gly Gly Asp Val Leu Phe Val Gly Lys646 His His Pro Leu His Asp Phe Ile Leu Glu Leu Tyr Gln Thr Gly661 Ser Thr Glu Pro Val Glu Val Pro Pro Glu Leu Cys His Gly Ile676 Gln Gly Lys Phe Ser Leu Asp Glu Glu Ala Ile Leu Pro Asp Gln691 Ile Val Cys Ser Pro Val Pro Met Leu Arg Asp Leu Thr Gln Asn706 Thr Val Val Ser Ile Asn Phe Lys Asp Pro Gln Leu Ala Glu Asp721 Tyr Ile Phe Lys Ala Val Met Leu Pro Gly Ala Arg Lys Pro Ala736 Ala Val Leu Lys Pro Ser Asp Trp Glu Lys Ser Ser Asn Gly Arg751 Gln Trp Lys Pro Gln Leu Gly Phe Asn Arg Asp Arg Arg Pro Val766 His Leu Asp Gln Ala Ala Phe Arg Thr Leu Gly His Val Met Pro781 Arg Gly Ser Gly Thr Gly Ile Tyr Ser Asn Ala Ala Pro Pro Pro796 Val Thr Tyr Gln Gly Asn Leu Tyr Arg Pro Leu Leu Arg Gly Gln811 Ala Gln Ile Pro Lys Leu Met Ser Asn Met Arg Pro Gln Asp Ser826 Trp Arg Gly Pro Pro Leu Leu Phe Gln Gln Gln Arg Phe Asp Arg841 Gly Val Gly Ala Glu Pro Leu Leu Pro Trp Asn Arg Met Leu Gln856 Thr Gln Asn Ala Ala Phe Gln Pro Asn Gln Tyr Gln Met Leu Ala871 Gly Pro Gly Gly Tyr Pro Pro Arg Arg Asp Asp Arg Gly Gly Arg886 Gln Gly Tyr Pro Arg Glu Gly Arg Lys Tyr Pro Leu Pro Pro Pro901 Ser Gly Arg Tyr Asn Trp Asn ( 4 ) SEQ ID NO:3 ( ⅰ )
(A) length: 22 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:GCCGTCTCTTTGGTTACGCTCG 22 (5) SEQ ID NO:4
(A) length: 22 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:TTTTAAAAGTTTAACTTTATTT 22 (6) SEQ ID NO:5
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:5:CCCCATATGATGGGAGTCCCGGCGTTCTTCCG 32 (7) SEQ ID NO:6
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:CCCCATATGATGGGAGTCCCGGCGTTCTTCCG 32 (8) SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 15 amino acid
(B) type: amino acid
(D) topological framework: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: SEQ ID NO:7:NH 2-Met-Gly-Val-Pro-Ala-Phe-Phe-Arg-Trp-Leu-Ser-Arg-Lys-Tyr-Pro-COOH

Claims (18)

1, a kind of isolated polypeptide-XRN-100 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.
2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.
3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.
4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:
(a) coding 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 9 5% homogenies (b) are arranged.
5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQID NO:2.
6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-3271 position among the sequence of 60-2783 position among the SEQ IDNO:1 or the SEQ ID NO:1.
7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.
8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:
(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or
(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.
9, a kind of preparation method with the active polypeptide of XRN-100 is characterized in that described method comprises:
(a) expressing under the XRN-100 condition, cultivate the described through engineering approaches host cell of claim 8;
(b) from culture, isolate and have the active polypeptide of XRN-100.
10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with XRN-100 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 XRN-100.
12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.
13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used to regulate XRN-100 in vivo, the method for external activity.
14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.
15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen the stand-in of XRN-100, agonist, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.
16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.
17,, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with XRN-100 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.
18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.
CN 99119819 1999-10-22 1999-10-22 Novel polypeptide-XRN-100 and polynucleotide coding said polypeptide Pending CN1303931A (en)

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