CN1308087A - Human testis specific protein gene coding protein - Google Patents

Abstract

The invention belongs to the field of human genome technology. The human testis-specific protein gene (testis-specific protein) has a full-length cDNA sequence of 2270 bp, an open reading frame of 1494 bp, and encodes 498 amino acids. The gene is in the Genbank (Genbank) The serial number is AY009105. The human testis-specific protein gene expression cloning of the present invention can be used to prepare a fusion protein, and the protein can be used to immunize animals to prepare monoclonal and polyclonal antibodies, and the gene clone can be used for the preparation of testis-specific function gene expression chips.

Description

Human testis specific protein gene coding protein

The invention belongs to the Human genome technical group field.

The white gene of human testicle specificity protein of the present invention is that the contriver obtains with the method for gene chip screening in reproductive medicine Jiangsu Province key lab of Nanjing Medical University, at first prepare people's testicular function gene expression chip, by comparing embryo and grownup's testicular function expression of gene, acquisition differential expression clone, prove full-length cDNA through sequencing, be 2270bp; Its open reading frame is 1494bp, its 498 amino acid of encoding.Consult gene pool, visible homologous EST fragment, and mostly be the testis expression.Because this gene is the adult testis high expression level, and the main difference of embryo and adult testis is the latter spermatogeny is arranged, therefore the white gene of finding of human testicle specificity protein is relevant with spermatogeny probably, use this gene clone, can be prepared into fusion rotein, with this protein immunization rabbit, can obtain mono-clonal and polyclonal antibody, this gene also can be used for preparing the testicular function gene expression chip.

Because the white gene of human testicle specificity protein still unmanned homologous gene in the Genbank database; the inventor all is initiative for the 26S Proteasome Structure and Function achievement in research of this gene. ( National Center for BiotechnologyInformation NCBI ) formally accepts the newcomer that this gene is Genbank, and accepting number is AY009105 in American National biotechnology information center.1 gggcacattg cagattgttc gggtaaattt aaaatgctag agcatgccct acgtgatgcc 61 aagatggcgg agacttgtat tgtgaaagaa aagcaagatt ataagcagaa attgaaggca 121 cttaagattg aagtcaacaa actaaaagag gacctcaatg aaaagacgac agaaaataat 181 gagcaacgag aagagatcat tcgcctcaag caagagaaaa gttgcctgca cgatgaattg 241 ctttttactg tagagagaga aaagaggaaa gatgaattgc ttaatattgc gaagtcaaag 301 caagaacgca caaattcaga actgcacaat ctgagacaga tttatgtaaa acaacagagt 361 gatctgcagt ttcttaattt caatgtggaa aattctcagg aattaataca gatgtatgac 421 tcaaagatgg aggaatcaaa ggctctggac tccagcagag acatgtgttt atcagacctt 481 gaaaataacc acccaaaagt cgatattaag agggaaaaaa atcagaagtc actgtttaag 541 gaccagaaat ttgaagccat gttggttcag caaaataggt cagacaagag ctcttgcgat 601 gaatgcaaag agaagaaaca acagatcgat actgtgtttg gggagaaaag tgtaattacg 661 ctgtcatcca tattcaccaa agacttagta gagaaacaca acctcccttg gtctctggga 721 ggaaaaaccc agattgaacc cgaaaacaaa attacattgt gcaagatcca cacaaaatca 781 ccaaaatgtc atggcactgg ggttcagaac gaaggaaaac aaccctcaga aacacccact 841 ttatctgatg agaagcagtg gcatgatgtc agtgtttacc tgggcctgac caactgtcca 901 agttcaaaac atccagaaaa gctggatgta gaatgtcaag atcagatgga aaggtccgaa 961 atctcatgct gccagaaaaa tgaagcctgt ctgggcgaaa gtggcatgtg tgactccaag1021 tgctgccacc cgagtaactt cataattgaa gccccaggcc acatgtctga cgtggagtgg1081 atgagtattt tcaagccttc caaaatgcag agaattgtcc gcctcaaatc tgggtgcacc1141 tgttcagaaa gcatctgtgg cacacaacat gactccccgg caagtgagct aattgccatc1201 caagattccc actctttggg ttcttcaaaa tctgccttga gagaagatga gacggagtcc1261 tcttccaata aaaagaactc acctacgagt ttgttaatct acaaagatgc accagcattc1321 aatgaaaagg cttcaattgt gttaccctcc caggatgatt tctcgcccac gagcaagctc1381 cagcgtttgc tggcggaatc tcgtcagatg gtgacggacc tggagctgaa cacactgctg1441 cccatcagcc atgagaatct cactggcagt gccacaaata tttctcatct atgtggaagg1501 cagaaagcag acaccaatac tgaatgaata cttaaccgta aaactgaaag aggattctag1561 ttcttcataa acggcactta attccagctg ggagcagaac tagaaagtta atttttaaac1621 atctacactt cattttcaag ttaaccattt ttgtgctgaa gaaatatttt catgtgtaag1681 aaagtagacc ttattgtaca tatagaaagt tggaattatg ctaagaatga aaaagacttc1741 tctgtaaaga tacagactac agttaaatgc tagagaagct ctttaaaaat gtgaatgtca1801 aatagagaaa gaacccctgc atagaaagtg ctgttttaac tatctgattt ttaaaaaatc1861 tgtgcataca tttaaattct aaacaatagc ttatcagagt cagctcaaaa tatatgagaa1921 acagtattct ctcatggttt tagcttttga ctttgctgtg taaatagaca taaggtgctt1981 tgatataaaa tataaagtgt aactggaaaa tagctcgagg tccttctgtc ccaagctgag2041 cagagcccca tctttctggg tctatattag tcccacctac tgacacaaac aaaagcttgc2101 tggaagatcg agttttagac gcatttttaa aaatcttaaa gactaaaaca cttccatttt2161 aacttgtaaa gtaatttaat tttttaaaga ttatactata tgcctctgtg tcttctctaa2221 aagaatagat caacttcagt ccataaaaga tatttttaat attaaagaaacDNAMLEHALRDAKMAETCIVKEKQDYKQKLKALKIEVNKLKEDLNEKTTENNEQREEIIRLKQEKSCLHDELLFTVEREKRKDELLNIAKSKQERTNSELHNLRQIYVKQQSDLQFLNFNVENSQELIQMYDSKMEESKALDSSRDMCLSDLENNHPKVDIKREKNQKSLFKDQKFEAMLVQQNRSDKSSCDECKEKKQQIDTVFGEKSVITLSSIFTKDLVEKHNLPWSLGGKTQIEPENKITLCKIHTKSPKCHGTGVQNEGKQPSETPTLSDEKQWHDVSVYLGLTNCPSSKHPEKLDVECQDQMERSEISCCQKNEACLGESGMCDSKCCHPSNFIIEAPGHMSDVEWMSIFKPSKMQRIVRLKSGCTCSESICGTQHDSPASELIAIQDSHSLGSSKSALREDETESSSNKKNSPTSLLIYKDAPAFNEKASIVLPSQDDFSPTSKLQRLLAESRQMVTDLELNTLLPISHENLTGSATNISHLCGRQKADTNTE

The white gene open reading frame of human testicle specificity protein aminoacid sequence

The objective of the invention is: in view of the white gene of human testicle specificity protein is the brand-new gene that the inventor finds, human at present understanding and the understanding to this gene only limits to the scientific research that the inventor does.Because this gene is the gene relevant with the testis specific function, and be the low expression of embryo, adult's high expression level, therefore this gene is relevant with intratesticular spermatogeny probably, its unconventionality expression may influence the male sex's reproductive function, therefore the research of this gene can be used for: 1. by this gene Fusion albumen of preparation, be used to study the relation of this gene and testicular function, also may become a kind of biologics for the treatment of the male testical relative disease.

2. prepare specific antibody: the specific antibody of gene coded protein is the indispensable important tool of research gene structure and function, the polyclone of the white gene coded protein of human testicle specificity protein and monoclonal antibody, can be used as immunohistochemical staining, ELISA, immuno-electron microscope, multiple detection method such as co-immunoprecipitation according to the immunology principle design, can be used for location and the quantitative examination of this gene coded protein in histocyte, also can be used for various biological specimens (as blood, seminal fluid, urine etc.) content research in and protein-protein interaction research etc.Therefore, this antibody might be used for preparation and the white abnormal gene expression relative disease of human testicle specificity protein diagnostic kit, and might be used to prepare antifertility drug.

3. prepare gene diagnosis chip: functional gene is used to prepare the importance that gene diagnosis chip is the gene development research, all significant at aspects such as medical diagnosis on disease (as male sterility, sexual disorder) and drug screenings.The proteic preparation of the white gene fusion of technical solution of the present invention 1. human testicle specificity proteins is with the nucleotide sequence and the plasmid pDEST of the white gene open reading frame of human testicle specificity protein ^TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, cultivate 0.5OD600 for 30 ℃ in the LB of no NaCl, and after 0.3MNaCl induced, ultrasonication obtained the fusion rotein of purifying after GST affinity chromatography column purification.2. the white gene coded protein specific polyclonal antibody of human testicle specificity protein

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), through the dialysis after again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) balanced mix, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, totally three times, after three months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity, measure antibody titers in each immunize rabbit serum.Screen high titre serum, further do competition and suppress the specificity that immunity test is determined antibody with antigen.Determine that according to ELISA result its dilution that is used for immunohistochemical staining multiplication number is 1: 200～500.3. the white gene coded protein monoclonal antibody specific of human testicle specificity protein

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) mixes, immunity Balb/C mouse (intraperitoneal injection), every other week once, continuous 2-3 time, vein is strengthened once before merging, after ELISA method confirmation production of antibodies, get the spleen separating Morr. cell, merge the generation hybridoma with the myeloma cell then.Positive colony is through subclone repeatedly, until the monoclonal hybridoma strain that obtains the white gene of the anti-human testicle specificity protein of secretion.4. the white gene of human testicle specificity protein is used to prepare the testicular function gene expression chip

(1) with the nucleotide sequence design primer of the white gene of human testicle specificity protein that obtains, the white gene order of human testicle specificity protein of amplification total length is as the sample of some film.

(2) with Britain BioRobotics put automatically the film instrument with specimens point on 8 * 12cm nylon membrane, 2 points of each specimens point, several approximately ng of every DNA amount. 8 housekeeping genes of positive control (a.ribosomal protein S9; B.Actin gamma; C.glyceraldehyde-3-phosphate dehydrogenase; D.hypoxanthinephosphoribosyltransferase 1; E.H.sapiens mRNA for 23KD highly basicprotein; F.ubiqnitin C; G.Phospholipase A2; H.ubiquitin carboxy I-ferminal esterase LI).Negative control adds lambda bacteriophage dna and P-blue plasmid.(3) chip that will contain this gene is used for the diagnosis and the drug screening of the white genetically deficient of human testicle specificity protein ³³The P mark:

Extracting sample mRNA to be checked or DNA are with Random Primer labelling method mark sample mRNA to be checked or DNA, as the probe of hybridization.Hybridization:

After the nylon membrane label probe hybridization that point is made, oven dry is shielded compressing tablet with phosphorus in the hybrid heater (68 ℃).Signal scanning and analysis:

Analyze hybridization signal with registering instrument.According to the white expression of gene protein level of strong and weak analyzing influence human testicle specificity protein of hybridization signal or the disappearance of the white gene of human testicle specificity protein.

Claims (4)

1.一种精子功能特异基因克隆制备的融合蛋白，其特征是人睾丸特异性蛋白基因全部开放阅读框氨基酸，序列为：MLEHALRDAKMAETCIVKEKQDYKQKLKALKIEVNKLKEDLNEKTTENNEQREEIIRLKQEKSCLHDELLFTVEREKRKDELLNIAKSKQERTNSELHNLRQIYVKQQSDLQFLNFNVENSQELIQMYDSKMEESKALDSSRDMCLSDLFNNHPKVDIKREKNQKSLFKDQKFEAMLVQQNRSDKSSCDECKEKKQQIDTVFGEKSVITLSSIFTKDLVEKHNLPWSLGGKTQIEPENKITLCKIHTKSPKCHGTGVQNEGKQPSETPTLSDEKQWHDVSVYLGLTNCPSSKHPEKLDVECQDQMERSEISCCQKNEACLGESGMCDSKCCHPSNFIIEAPGHMSDVEWMSIFKPSKMQRIVRLKSGCTCSESICGTQHDSPASELIAIQDSHSLGSSKSALREDETESSSNKKNSPTSLLIYKDAPAFNEKASIVLPSQDDFSPTSKLQRLLAESRQMVTDLELNTILPISHENLTGSATNISHLCGRQKADTNTE 2. According to claim 1, the polyclonal antibody prepared against the amino acid sequence of the open reading frame of the human testis-specific protein gene is characterized in that after the fusion protein is mixed with complete and incomplete adjuvants, the polyclonal antibody is prepared by immunizing New Zealand rabbits. 3. The monoclonal antibody prepared against the open reading frame amino acid sequence of the human testis-specific protein gene according to claim 1 is characterized in that the fusion protein is mixed with complete and incomplete adjuvants, immune Balb/C mice, and isolated spleen B lymphocytes And fused with myeloma cells to produce hybridoma cells, and further screen the monoclonal secreting IgG antibody by subcloning method, ELISA and antibody typing method to prepare monoclonal antibody. 4. According to claim 1, the open reading frame amino acid sequence of the human testis-specific protein gene is used to prepare a testis functional gene expression chip, which is characterized in that the cDNA of the human testis-specific protein gene is amplified by PCR and spotted on the chip for chip preparation.