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

Abstract

The invention belongs to the field of human genome technology. The full-length cDNA sequence of the human testis specific protein kinase gene (NYD-SPK) gene is 3617 bp, its open reading frame sequence is 2991 bp, and it encodes 966 Amino acid, the number of this gene in Genbank (Genbank) is AF332194. The present invention uses NYD-SPK gene cloning to prepare fusion protein, and the protein can be used to immunize animals to prepare monoclonal and polyclonal antibodies. At the same time, gene cloning can be used for the preparation of testicular specific function gene expression chips, and the expressed protein may also be used to treat related diseases. gene medicines.

Description

human testis specific protein kinase gene encoding protein

The invention belongs to the technical field of human genome.

The human testis-specific protein kinase gene (testis specific protein kinase gene, NYD-SPK) of the present invention was obtained by the inventor in the Key Laboratory of Reproductive Medicine, Jiangsu Province, Nanjing Medical University, through gene chip method screening, and first prepared a human testis functional gene expression chip , By comparing the expression of embryonic and adult testis functional genes, a differentially expressed clone was obtained, which was proved to be a full-length cDNA of 3617 bp by sequence determination; its open reading frame was 2991 bp, and it encoded 996 amino acids. Check the gene bank, the gene is a new gene, has tyrosine, serine phosphorylation kinase activity. Since this gene is highly expressed in adult testis, the human testis-specific protein kinase gene discovered may be related to testis development. This gene clone can be used to prepare a fusion protein, and the protein can be used to immunize rabbits to obtain monoclonal and polyclonal antibodies. , the gene can also be used to prepare testis functional gene expression chip. Fusion proteins may also become gene medicines for treating related diseases.

Since the human testis-specific protein kinase gene has no homologous gene in the Genbank database, the inventors' research results on the structure and function of the gene are all groundbreaking.美国国家生物技术情报中心(National Center for BiotechnologyInformation,NCBI)已正式接受该基因为Genbank的新成员，接受号为AF332194。1 ggaaaagtgt gaggtcagta gtagtttttg tggtacgtgc agtggtggcc gcttcaagga61 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

人睾丸激酶基因cDNA序列MDEDTHYDKVEDVVGSHIEDAVTFWAQSINRNKDIMKIGCSLSEVCPQASSVLGNLDPNKIYGGLFSEDQCWYRCKVLKIISVEKCLVRYIDYGNTEILNRSDIVEIPLELQFSSVAKKYKLWGLHIPSDQEVTQFDQGTTFLGSLIFEKEIKMRIKATSEDGTVIAQAEYGSVDIGEEVLKKGFAEKCRLASRTDICEEKKLDPGQLVLRNLKSPIPLWGHRSNQSTFSRPKGHLSEKMTLDLKDENDAGNLITFPKESLAVGDFNLGSNVSLEKFKQDQKLIEENEKLKTEKDALLESYKALELKVEQIAQELQQEKAAAVDLTNHLEYTLKTYIDTRMKNLAAKMEILKEMRHVDISVRFGKDLSDAIQVLDEGCFTTPASLNGLEIIWAEYSLAQENIKTCEYVSEGNILIAQRNEMQQKLYMSVEDFILEVDESSLNKRLKTLQDLSVSLEAVYGQAKEGANSDEILKKFYDWKCDKREEYTSVRSETDASLHRLVAWFQRTLKVFDLSVEGSLISEDAMDNIDEILEKTESSVCKELEIALVDQGDADKEIISNTYSQVLQKIHSEERLIATVQAKYKDSIEFKKQLIEYLNKSPSYDHLLSIKKTLKSLKALLRWKLVEKSNLEESDDPDGSQIEKIKEEITQLRNNVFQEIYHEREEYEMLTSLAQKWFPELPLLHPEIGLLKCMNSGGLLTMSLERDLLDAEPMKELSSKRPLVRSEVNGQIILLKGYSVDVDTEAKVIERAATYHRAWREAEGDSGLLPLIFLFLCKSDPMAYLMVPYYPRANLNAAQANMPLNSEETLKVMKGVAQGLHTLHKADIIHGSLHQNNVFALNREQGIVGDFDFTKSVSQRASVNMMVGDLSLMSPELKMGKPASPGSDLYAYGCLLLWLSVQNQEFEINKDGIPKYDQFHLDDKVISLLCSLICYRSSMTGEQVLNAECFLMPKEQSVPNPEKDTEYTLYKKEEEIKPENLDKCMEKTRNGEANFDC

The open reading frame amino acid sequence of human testis-specific protein kinase gene

The purpose of the present invention is: in view of the fact that the human testis-specific protein kinase gene is a brand-new gene discovered by the inventor, the current human knowledge and understanding of this gene is limited to the scientific research work done by the inventor. Since this gene is related to the specific function of the testis, and it is low expressed in the embryo and highly expressed in the adult, it is likely to be related to the development of the testis, and its abnormal expression may affect male reproductive function, so the study of this gene can be used for: ① By preparing the fusion protein of the gene, it is used to study the relationship between the gene and testis function, and it may also become a biological drug for treating male testis-related diseases.

② Preparation of specific antibodies: specific antibodies for gene-encoded proteins are an indispensable and important tool for studying gene structure and function. Polyclonal and monoclonal antibodies for proteins encoded by the human testis protein kinase gene can be used for immunohistochemical staining, ELISA , immunoelectron microscopy, immunoprecipitation and other detection methods designed according to immunological principles can be used for localization and quantitative research on the protein encoded by the gene in tissue cells, and can also be used for various biological samples (such as blood, semen, urine, etc.) etc.), research on content changes and protein-protein interaction research, etc. Therefore, the antibody may be used in the preparation of a diagnostic kit for diseases related to the abnormal expression of the human testis-specific protein kinase gene, and may be used in the preparation of antifertility drugs.

③Preparation of genetic diagnostic chips: The use of functional genes in the preparation of genetic diagnostic chips is an important aspect of gene development research, and is of great significance in disease diagnosis (such as male infertility, low sexual function) and drug screening. Technical solution of the present invention 1. Preparation of Human Testis-Specific Protein Kinase Gene Fusion Protein

The nucleotide sequence of the open reading frame of the human testis-specific protein kinase gene and the plasmid pDEST ^TM 15 were used to prepare the GST-SP2-pDEST15 expression plasmid, cultured in NaCl-free LB at 30°C to 0.5 OD600, and induced by 0.3M NaCl , purified by GST affinity chromatography column after ultrasonic pulverization to obtain purified fusion protein. 2. Human testis-specific protein kinase gene-encoded protein-specific polyclonal antibody

Combine the fusion protein with the carrier protein KLH (Keyhole Limpet Hymocyanin), and then mix it with an equal amount of complete/incomplete adjuvant (Complete/Incomplete Freund's Adiuvant) after dialysis, and immunize New Zealand rabbits (subcutaneous injection in the back), every 3~ Inject once every 4 weeks for a total of three times, and blood was collected three months later to separate serum. ELISA was performed with the animal serum before immunization as a control, and the antibody titer in each immunized rabbit serum was determined. Screen high-titer sera, and further use the antigen as a competitive inhibition immunoassay to determine the specificity of the antibody. According to the results of ELISA, the dilution doublings for immunohistochemical staining were determined to be 1:200-500.3. Human testis-specific protein kinase gene-encoded protein-specific monoclonal antibody

Combine the fusion protein with the carrier protein KLH (Keyhole Limpet Hymocyanin), and then mix it with complete/incomplete adjuvant (Complete/Incomplete Freund's Adiuvant) after dialysis, and immunize Balb/C mice (intraperitoneal injection), once every other week , 2-3 times in a row, and the vein was strengthened once before fusion. After confirming the production of antibodies by ELISA method, the spleen was taken to separate spleen cells, and then fused with myeloma cells to produce hybridoma cells. Positive clones were subcloned several times until a monoclonal hybridoma cell line secreting anti-human testis-specific protein kinase antibody was obtained. 4. Human testis-specific protein kinase gene used to prepare testis functional gene expression chip

(1) Design primers with the obtained nucleotide sequence of the human testis protein kinase gene, amplify the full-length human testis protein kinase gene sequence, and use it as a sample for spotting the membrane. (2) Use the British BioRobotics automatic film spotting instrument to spot the specimen on an 8×12 cm nylon membrane, 2 spots for each sample, and the amount of DNA in each spot is about a few ng. ; positive control 8 housekeeping genes (a.ribosomal protein S9; b.Actin gamma; c.glyceraldehyde-3-phosphate dehydrogenase; d.hypoxanthinephosphoribosyltransferase 1; eHsapiens mRNA for 23KD highly basicprotein; f.ubiqnitin C; g.Phospholipase A2; h. ubiquitin carboxy I-ferminal esterase L I). Negative control plus lambda phage DNA and P-blue plasmid. (3) The chip containing this gene is used for the diagnosis and drug screening of human testis-specific protein kinase gene deletion ³³ p marker:

Extract the mRNA or DNA of the sample to be tested, and use the Random Primer labeling method to label the mRNA or DNA of the sample to be tested as a probe for hybridization.

hybridization:

After hybridizing the prepared nylon membrane-labeled probes, dry them in a hybridization oven (68° C.), and press them with a phosphor screen. Signal Scanning and Analysis:

Hybridization signals were analyzed with a recorder. According to the strength analysis of the hybridization signal, the expression level of human testis kinase gene protein or the deletion of human testis specific protein kinase gene is affected.

Claims (5)

1. The fusion protein prepared by cloning the human testis-specific protein kinase gene (NYD-SPK), its specific 征是NYD-SPK基因全部开放阅读框氨基酸，序列为：MDEDTHYDKVEDVVGSHIEDAVTFWAQSINRNKDIMKIGCSLSEVCPQASSVLGNLDPNKIYGGLFSEDQCWYRCKVLKIISVEKCLVRYIDYGNTEILNRSDIVEIPLELQFSSVAKKYKLWGLHIPSDQEVTQFDQGTTFLGSLIFEKEIKMRIKATSEDGTVIAQAEYGSVDIGEEVLKKGFAEKCRLASRTDICEEKKLDPGQLVLRNLKSPIPLWGHRSNQSTFSRPKGHLSEKMTLDLKDENDAGNLITFPKESLAVGDFNLGSNVSLEKFKQDQKLIEENEKLKTEKDALLESYKALELKVEQIAQELQQEKAAAVDLTNHLEYTLKTYIDTRMKNLAAKMEILKEMRHVDISVRFGKDLSDAIQVLDEGCFTTPASLNGLEIIWAEYSLAQENIKTCEYVSEGNILIAQRNEMQQKLYMSVEDFILEVDESSLNKRLKTLQDLSVSLEAVYGQAKEGANSDEILKKFYDWKCDKREEYTSVRSETDASLHRLVAWFQRTLKVFDLSVEGSLISEDAMDNIDEILEKTESSVCKELEIALVDQGDADKEIISNTYSQVLQKIHSEERLIATVQAKYKDSIEFKKQLIEYLNKSPSVDHLLSIKKTLKSLKALLRWKLVEKSNLEESDDPDGSQIEKIKEEITQLRNNVFQEIYHEREEYEMLTSLAQKWFPELPLLHPEIGLLKCMNSGGLLTMSLERDLLDAEPMKELSSKRPLVRSEVNGQIILLKGYSVDVDTEAKVIERAATYHRAWREAEGDSGLLPLIFLFLCKSDPMAYLMVPYYPRANLNAAQANMPLNSEETLKVMKGVAQGLHTLHKADIIHGSLHQNNVFALNREQGIVGDFDFTKSVSQRASVNMMVGDLSLMSPELKMGKPASPGSDLYAYGCLLLWLSVQNQEFEINKDGIPKVDQFHLDDKVISLLCSLICYRSSMTGEQVLNAECFLMPKEQSVPNPEKDTEYTLYKKEEEIKPENLDKCMEKTRNGEANFDC 2. According to the polyclonal antibody prepared against the amino acid sequence of the open reading frame of NYD-SPK gene according to claim 1, it 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. According to the monoclonal antibody prepared against the amino acid sequence of the open reading frame of NYD-SPK gene according to claim 1, it is characterized in that the fusion protein is mixed with complete and incomplete adjuvant, immunized Balb/C mice, isolated spleen B lymphocytes and Myeloma cells are fused to produce hybridoma cells, and the monoclonal secreting IgG antibody is further screened by subcloning method, ELISA and antibody typing method to prepare monoclonal antibody. 4. According to claim 1, the amino acid sequence of the open reading frame of NYD-SPK gene is used to prepare a testis functional gene expression chip, which is characterized in that the NYD-SPK gene cDNA is amplified by PCR and spotted on the chip for chip preparation. 5. According to claim 1, the fusion protein is prepared for the amino acid sequence of the open reading frame of the NYD-SPK gene, which is characterized in that the fusion protein can be used as a gene drug for the preparation of related diseases.