CN1319660A - Human testis specific protein kinase gene coding protein - Google Patents

Abstract

The present invention belongs to the field of human genome technology, it desciribes human testis specific protein kinase gene, NYD-SPK, its gene total length cDNA sequence is 3617 bp, its open reading frame sequence is 2991 bp, it codes 966 amino acids, and the serial number of said gene in bank is AF332194. Said invention utilizes NYD-SPK gene clone to prepare fusion protein, and can use said protein to immunize animal to prepare mojnoclonal antibody and polyclonal antibody, at the same time the gene clone can be usexed for preparing testis specific function gene expression chip, and said expression protein also can be made into gene medicine for curing related diseases.

Description

The protein kinase gene coded protein of human testis specific

The invention belongs to the Human genome technical group field.

The protein kinase gene of human testis specific of the present invention (testis specificprotein kinase gene, NYD-SPK) be 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, proving full-length cDNA through sequencing, is 3617 bp; Its open reading frame is 2991 bp, its 996 amino acid of encoding.Consult gene pool, this gene is new gene, has tyrosine, the serine phosphorylation kinase activity.Because this gene is the adult testis high expression level, therefore the protein kinase gene of the human testis specific of finding may be relevant with testicualr development, can be prepared into fusion rotein with this gene clone, 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.Fusion rotein also may become the genomic medicine of treatment relative disease.

Because human testis specific protein kinase gene 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 AF332194 in American National biotechnology information center.1?ggaaaagtgt?gaggtcagta?gtagtttttg?tggtacgtgc?agtggtggcc?gcttcaagga?61?ctattttcgt?cgctgcttgc?gttttaagtc?tccaatcctg?ggacgaggcc?catttcaggg121?ttttctgtca?ccttctagcc?gcccgccacc?ccagaggggt?gccagatgcc?ccagtttttg181?agtgcatgca?ggcaggcgaa?agtggctcga?aaagacctcc?ggcctgaatg?atctccatga241?agacgcggtc?acgcagcctc?cacactctct?tccccttctc?gaaaattctg?tgccatttaa301?gtttcaaaat?cccttataaa?ataagtgtca?gagagctacg?tgtacccgtt?tcttccaaat361?tttcatcacg?aagtggagat?ttggacgtac?tattttgtga?tctctgtttg?cagaaagtgg421?gtacggcatc?agtgccttca?taatcaggtt?tttcaggaat?tgttcagatg?gatgaagata481?cacattacga?taaagtggaa?gatgtggttg?gaagtcacat?agaagatgca?gtaacatttt541?gggcccagag?tatcaataga?aataaggata?tcatgaagat?tggttgctca?ctgtctgaag601?tttgccccca?ggccagttca?gttttgggga?atcttgaccc?aaacaagatt?tatggtggat661?tattttctga?agatcagtgt?tggtacagat?gcaaagtact?gaaaatcatc?agcgttgaaa721?agtgtctggt?gaggtacatt?gactatggaa?atactgaaat?tctaaatcga?tctgatatag781?ttgaaattcc?tttggagctg?cagttttcta?gtgttgccaa?aaagtataaa?ctttggggac841?tacacattcc?ttctgatcaa?gaagttaccc?agtttgatca?gggcacaacc?tttttgggga901?gcttgatttt?tgaaaaggaa?ataaaaatga?gaattaaagc?aacctctgaa?gatggaacag961?ttattgctca?ggctgagtat?ggcagtgtgg?atatagggga?agaggtgctt?aagaaaggat1021?ttgcagagaa?atgcagactt?gcttccagaa?ctgacatctg?tgaggaaaaa?aaattggatc1081?ctggtcaact?tgttctcagg?aacctcaaaa?gccccattcc?tttgtggggg?catagatcaa1141?accagtcaac?cttcagcagg?cccaaggggc?acttaagtga?gaaaatgact?cttgacttga1201?aggatgaaaa?tgatgcaggc?aatcttataa?catttccaaa?ggaaagtttg?gctgttggtg1261?actttaattt?agggtctaac?gtcagcctgg?aaaaatttaa?gcaggaccag?aaactgattg1321?aagaaaatga?aaaacttaaa?acagagaagg?acgctcttct?tgaaagttat?aaggcgttag1381?aattgaaagt?agagcagatt?gcccaggagc?tgcagcaaga?gaaggcagct?gctgtggatt1441?tgactaacca?cttagaatac?actctgaaga?cctatataga?taccagaatg?aaaaatctgg1501?cagctaagat?ggaaatactg?aaagaaatga?ggcatgtcga?catcagtgtc?cgtttcggaa1561?aagacctttc?agatgctata?caagtgttgg?atgaagggtg?ctttactact?ccagcttctt1621?tgaacggatt?agagataata?tgggcagaat?acagtctggc?tcaggagaat?attaaaactt1681?gtgaatatgt?gagtgaaggg?aatattttga?ttgcccaaag?aaatgaaatg?cagcagaagc1741?tgtacatgtc?agtagaagat?tttattctgg?aagttgatga?gtcatctctt?aataaacgct1801?taaaaacatt?gcaggatttg?tcagtctctt?tagaagcagt?gtatggacaa?gccaaagaag1861?gagcaaattc?tgatgaaata?cttaaaaaat?tttatgactg?gaagtgtgat?aaaagagagg1921?agtacaccag?tgttagaagt?gaaacagacg?cttctctgca?ccgtcttgta?gcatggttcc1981?aaagaacctt?aaaggttttt?gacctatctg?tggaaggatc?actgatttca?gaagacgcaa2041?tggataatat?tgatgaaatc?ctagagaaga?ctgagtcaag?tgtctgcaaa?gagctggaga2101?tagctctggt?tgatcaaggt?gatgcagaca?aggagataat?ttcaaataca?tatagtcaag2161?tactgcaaaa?gattcattca?gaggaaaggc?tcattgccac?agtacaagct?aagtacaagg2221?acagtattga?gtttaaaaag?cagcttattg?aatatttaaa?taagagtccc?agtgtggatc2281?acttgctatc?cattaagaag?acattgaaaa?gcttaaaagc?tctactcaga?tggaaattgg2341?ttgaaaagag?taatttggaa?gagtcagatg?atcctgatgg?ctctcaaatt?gagaaaataa2401?aagaagaaat?aactcagctg?cgcaataatg?tctttcagga?aatttatcat?gagagagagg2461?aatatgagat?gctaactagt?ttggcacaga?aatggttccc?tgagctgcct?ctgcttcatc2521?ctgaaatagg?attactcaaa?tgcatgaact?ctggtggtct?ccttacaatg?agcttggaac2581?gagatcttct?tgatgctgag?cccatgaagg?aacttagcag?caagcgtcct?ttggtacgtt2641?ctgaggttaa?tgggcagata?attctgttaa?agggctattc?tgtggatgtt?gacacagaag2701?ccaaggtgat?tgagagagca?gccacctacc?atagagcttg?gagagaagct?gaaggagact2761?cagggttact?gccattgata?ttcctgtttt?tatgtaagtc?tgatcctatg?gcttatctga2821?tggtcccata?ctaccctagg?gcaaacctga?atgctgctca?agccaacatg?cctttaaatt2881?cagaagaaac?tttaaaggtc?atgaaaggtg?ttgcccaggg?tctgcataca?ttgcataagg2941?ctgacataat?tcatggatca?cttcatcaga?acaatgtatt?tgctttaaac?cgtgaacaag3001?gaattgttgg?agattttgac?ttcaccaaat?ctgtgagtca?gcgagcctcg?gtgaacatga3061?tggttggtga?cttgagtttg?atgtcacctg?agttgaaaat?gggaaaacct?gcttctccag3121?gttcagactt?atatgcttat?ggctgcctct?tattatggct?ttctgttcaa?aatcaggagt3181?ttgagataaa?taaagatgga?atccccaaag?tggatcagtt?tcatttggat?gataaagtca3241?tatccctcct?ctgtagcttg?atatgttata?gaagttcaat?gactggtgaa?caagttttaa3301?atgctgaatg?tttcttgatg?ccaaaggagc?aatcagttcc?aaacccagaa?aaagatactg3361?aatacaccct?atataaaaag?gaagaagaaa?taaagccgga?gaatttggat?aaatgtatgg3421?agaagacaag?aaatggtgaa?gccaactttg?attgttaaat?tattattgtt?gttgttgcaa3481?aggttctttt?taaaaacttt?gtttggtttg?gttaatacac?agaaatatct?agaaatgttc3541?tgggactagt?tgagttgtat?ctttagtatt?caggttgtga?aaaataaaga?tgtttggcta3601?tgcgcttaaa?aaaaaaa

cDNAMDEDTHYDKVEDVVGSHIEDAVTFWAQSINRNKDIMKIGCSLSEVCPQASSVLGNLDPNKIYGGLFSEDQCWYRCKVLKIISVEKCLVRYIDYGNTEILNRSDIVEIPLELQFSSVAKKYKLWGLHIPSDQEVTQFDQGTTFLGSLIFEKEIKMRIKATSEDGTVIAQAEYGSVDIGEEVLKKGFAEKCRLASRTDICEEKKLDPGQLVLRNLKSPIPLWGHRSNQSTFSRPKGHLSEKMTLDLKDENDAGNLITFPKESLAVGDFNLGSNVSLEKFKQDQKLIEENEKLKTEKDALLESYKALELKVEQIAQELQQEKAAAVDLTNHLEYTLKTYIDTRMKNLAAKMEILKEMRHVDISVRFGKDLSDAIQVLDEGCFTTPASLNGLEIIWAEYSLAQENIKTCEYVSEGNILIAQRNEMQQKLYMSVEDFILEVDESSLNKRLKTLQDLSVSLEAVYGQAKEGANSDEILKKFYDWKCDKREEYTSVRSETDASLHRLVAWFQRTLKVFDLSVEGSLISEDAMDNIDEILEKTESSVCKELEIALVDQGDADKEIISNTYSQVLQKIHSEERLIATVQAKYKDSIEFKKQLIEYLNKSPSYDHLLSIKKTLKSLKALLRWKLVEKSNLEESDDPDGSQIEKIKEEITQLRNNVFQEIYHEREEYEMLTSLAQKWFPELPLLHPEIGLLKCMNSGGLLTMSLERDLLDAEPMKELSSKRPLVRSEVNGQIILLKGYSVDVDTEAKVIERAATYHRAWREAEGDSGLLPLIFLFLCKSDPMAYLMVPYYPRANLNAAQANMPLNSEETLKVMKGVAQGLHTLHKADIIHGSLHQNNVFALNREQGIVGDFDFTKSVSQRASVNMMVGDLSLMSPELKMGKPASPGSDLYAYGCLLLWLSVQNQEFEINKDGIPKYDQFHLDDKVISLLCSLICYRSSMTGEQVLNAECFLMPKEQSVPNPEKDTEYTLYKKEEEIKPENLDKCMEKTRNGEANFDC

The protein kinase gene open reading frame aminoacid sequence of human testis specific

The objective of the invention is: in view of the protein kinase gene of human testis specific 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 testicualr development 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 PROTEINKINASE 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 human testis specific protein kinase gene abnormal expression relative disease 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 preparation of the protein kinase gene fusion rotein of technical solution of the present invention 1. human testis specifics

Nucleotide sequence and plasmid pDEST with the protein kinase gene open reading frame of human testis specific ^TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, cultivate 0.5 OD600 for 30 ℃ in the LB of no NaCl, and after 0.3M NaCl induced, ultrasonication obtained the fusion rotein of purifying after GST affinity chromatography column purification.2. the protein kinase gene coded protein specific polyclonal antibody of human testis specific

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), through the dialysis after again with fully/Freund (Complete/Incomplete Freund ' sAdiuvant) 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 protein kinase gene coded protein monoclonal antibody specific of human testis specific

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdiuvant) 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 of the protein kinase antibody that obtains the anti-human testis specific of secretion.4. the protein kinase gene of human testis specific is used to prepare the testicular function gene expression chip

(1) with the nucleotide sequence design primer of people's testis protein kinase gene of obtaining, people's testis protein kinase gene sequence 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 L I).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 protein kinase gene disappearance of human testis specific 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.Disappearance according to the protein kinase gene of the strong and weak analyzing influence people testis kinase gene protein expression level of hybridization signal or human testis specific.

Claims (5)

1. human testis specific protein kinase gene (NYD-SPK) is cloned the fusion rotein of preparation, its spy

NYD-SPK,：MDEDTHYDKVEDVVGSHIEDAVTFWAQSINRNKDIMKIGCSLSEVCPQASSVLGNLDPNKIYGGLFSEDQCWYRCKVLKIISVEKCLVRYIDYGNTEILNRSDIVEIPLELQFSSVAKKYKLWGLHIPSDQEVTQFDQGTTFLGSLIFEKEIKMRIKATSEDGTVIAQAEYGSVDIGEEVLKKGFAEKCRLASRTDICEEKKLDPGQLVLRNLKSPIPLWGHRSNQSTFSRPKGHLSEKMTLDLKDENDAGNLITFPKESLAVGDFNLGSNVSLEKFKQDQKLIEENEKLKTEKDALLESYKALELKVEQIAQELQQEKAAAVDLTNHLEYTLKTYIDTRMKNLAAKMEILKEMRHVDISVRFGKDLSDAIQVLDEGCFTTPASLNGLEIIWAEYSLAQENIKTCEYVSEGNILIAQRNEMQQKLYMSVEDFILEVDESSLNKRLKTLQDLSVSLEAVYGQAKEGANSDEILKKFYDWKCDKREEYTSVRSETDASLHRLVAWFQRTLKVFDLSVEGSLISEDAMDNIDEILEKTESSVCKELEIALVDQGDADKEIISNTYSQVLQKIHSEERLIATVQAKYKDSIEFKKQLIEYLNKSPSVDHLLSIKKTLKSLKALLRWKLVEKSNLEESDDPDGSQIEKIKEEITQLRNNVFQEIYHEREEYEMLTSLAQKWFPELPLLHPEIGLLKCMNSGGLLTMSLERDLLDAEPMKELSSKRPLVRSEVNGQIILLKGYSVDVDTEAKVIERAATYHRAWREAEGDSGLLPLIFLFLCKSDPMAYLMVPYYPRANLNAAQANMPLNSEETLKVMKGVAQGLHTLHKADIIHGSLHQNNVFALNREQGIVGDFDFTKSVSQRASVNMMVGDLSLMSPELKMGKPASPGSDLYAYGCLLLWLSVQNQEFEINKDGIPKVDQFHLDDKVISLLCSLICYRSSMTGEQVLNAECFLMPKEQSVPNPEKDTEYTLYKKEEEIKPENLDKCMEKTRNGEANFDC

2. according to the described polyclonal antibody at the preparation of NYD-SPK gene open reading frame aminoacid sequence of claim 1, after it is characterized in that fusion rotein and complete and Freund mixing, immune new zealand rabbit prepares polyclonal antibody.

3. according to the described monoclonal antibody of claim 1 at the preparation of NYD-SPK gene open reading frame aminoacid sequence, it is characterized in that fusion rotein mixes with complete and Freund, immunity Balb/C mouse, the separating spleen bone-marrow-derived lymphocyte and and the myeloma cell merge the generation hybridoma, further, prepare monoclonal antibody with the mono-clonal of subclone method, ELISA and antibody typing method screening IgG secretion antibody.

4. it is characterized in that pcr amplification NYD-SPK gene cDNA according to claim 1 is described at NYD-SPK gene open reading frame aminoacid sequence is used to prepare the testicular function gene expression chip, put in chip and be used for chip preparation.

5. according to claim 1, prepare fusion rotein at NYD-SPK gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the genomic medicine of preparation treatment relative disease.