CN1316434A - Human specific protein kinase-15 gene coding protein - Google Patents

Abstract

The invention belongs to the field of human genome technology. The full-length cDNA sequence of the human specific protein kinase-15 gene (specific protein kinase 15, NYD-SP15) is 3364 bp, its open reading frame sequence is 1545 bp, and it encodes 514 Amino acid, the number of this gene in Genbank (Genbank) is AY027525. The present invention utilizes NYD-SP15 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 testis-specific function gene expression chips, and the expressed protein may also be used to treat related diseases. gene medicines.

Description

Human specific protein kinase-15 gene encoding protein

The invention belongs to the technical field of human genome.

The human-specific protein kinase-15 gene (specific protein kinase 15, NYD-SP15) of the present invention was obtained by the inventor in the Key Laboratory of Reproductive Medicine, Jiangsu Province, Nanjing Medical University, by gene chip method screening, and first prepared human testis functional gene expression Chip, by comparing the expression of embryonic and adult testis functional genes, differentially expressed clones were obtained, which were proved to be full-length cDNA of 3364bp by sequence determination; its open reading frame was 1545bp, and its encoding was 514 amino acids. Check out GeneBank, the gene for the new gene. Since this gene is highly expressed in adult testes, the human-specific protein kinase-15 gene discovered may be related to testis development. The 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 a testis functional gene expression chip. Fusion proteins may also become gene medicines for treating related diseases.

Since the human-specific protein kinase-15 gene has no homologous gene in the Genbank database, the inventors' research results on the structure and function of this gene are all groundbreaking.美国国家生物技术情报中心(National Center for BrotechnologyInformation,NCBI)已正式接受该基因为Genbank的新成员，接受号为AY027525。1 gggggggggg cgctagggcc gagatcatgt ctgactggga gaggtttcct tggcagcaga61 ggacgctagg tttgggatga aagaagctgg gcagatgcaa aatctggaga gcgcgagggc121 cgggcggtca gtcagcaccc agactggcag catgaccggt cagataccaa ggctttctaa181 agtcaacctt ttcactctgc tcagcctctg gatggagctc tttccagcag aagcccagcg241 gcaaaaatct cagaaaaatg aagagggaaa gcatggaccc ttaggagata atgaagagag301 gaccagagta tctactgaca aaagacaggt aaagagaact ggtcttgtgg tggtgaaaaa361 catgaaaatt gttggtctcc actgttctag tgaagattta catgccgggc agattgctct421 tattaaacat gggtcaaggc tgaaaaactg tgatctttat ttttccagaa aaccatgttc481 tgcttgtttg aaaatgattg taaatgctgg agttaaccga atttcatact ggcctgctga541 tccagaaata agtttgctta cggaggcttc tagttctgaa gatgcaaagt tagatgccaa601 agcagtggaa agattgaagt caaacagtcg ggcccatgtg tgtgtcttac ttcaaccttt661 ggtgtgttat atggtgcagt ttgtagagga gacctcttac aaatgtgact ttattcaaaa721 aattacaaaa acattgccgg atgctaacac tgacttttat tatgaatgta aacaagaaag781 aataaaagaa tatgaaatgt tatttttggt ttcaaatgaa gaaatgcata agcaaatact841 gatgactata ggtttggaga acctgtgtga aaatccatac tttagcaatc taaggcaaaa901 catgaaagac cttatcctac ttttggccac agtagcttcc agtgtgccga actttaaaca961 cttcggattt taccgtagca atccagaaca gattaatgaa attcacaatc aaagtttgcc1021 acaggaaatt gcaaggcact gcatggttca ggccaggtta ttggcatatc gaactgagga1081 tcataaaaca ggagttgggg cagtcatttg ggcagaaggg aaatctagaa gttgtgatgg1141 aacaggtgcc atgtactttg taggatgtgg ttacaatgct tttcctgttg gatctgagta1201 tgctgacttc ccacacatgg atgacaagca gaaagacaga gaaataagga aattcagata1261 catcatacat gcggaacaga atgccttgac atttaggtgt caagaaataa aaccagaaga1321 aagaagcatg atttttgtga caaagtgccc atgtgatgag tgtgtacctt taattaaagg1381 tgcaggcata aaacaaatct atgcaggaga tgtagatgtt ggaaaaaaga aggcagacat1441 ctcttacatg aggttcgggg agcttgaagg tgttagcaaa tttacgtggc agctgaatcc1501 atcaggagct tatggtcttg aacaaaatga gcctgaaagg agagaaaatg gtgtgttgag1561 acctgtccca cagaaggaag agcagcacca ggacaagaag ctgcgcctcg gaatccacta1621 agaaggcctg tctacactgc aggggaacct caagaacttt tcattgcact tctaaaattc1681 agccttgttc attcagaaaa taaggatgga ttttgtgaat aattgaaaag attttttaag1741 gaagctaatt tatttctagg atacaaatgg tgttaataag aatatttgtc ttttagattt1801 atgtgtgggt tttccataat gtagtagtgt gttattttat tacacgaaat gagggagcaa1861 taacttcaac caatgtaatc accaacaggg actgatttct atttatcttg actttatatt1921 agccaatttg aaagggtctg cttcacaaga ggtcatgcct ctgcagggaa tcacacacct1981 tttgagaaat attaattctt ttgaattaca agagtgacag ttagtaaact gctccaggga2041 aataagtgtc atcttttaag agtcagtgta tagcaaacca aaagatcaca tttataccca2101 aagctttcta gtaatcagaa tttacttaat tagaattgac tcataaaaat aaagttagtg2161 gactttgtct ccatttttaa agtatatttt gtaaatgtta attctgtggt cctgtttatc2221 acagactttg ggtagcaata ggaagagagt gttattctga gacagtgtcg ctgcttgata2281 tcactgactc ttgaaatcac taacagtgaa cctcaaattt ggttatgatg ttaacagaga2341 agaaatattt gagtctggaa tattgaaact agctttccta gaattccatt aataaatgct2401 gctcccccat atagtcttcc cttctgtata attaacatag gttgtctcat ggctttgatc2461 ttcaaaagga agagcctttt aaaaacattt gccaacctca agaataaata ctgaaagtct2521 tgaaagtatg gtcatttcag ctattccaga ttgctgtctg tgatttctca catttttatt2581 tgctgagaaa cattcagaaa attattccaa aaatgaaggt acattttctg ttctctccta2641 ggttagagag gggtggcctg gggtgcttca tgagaaatgt cttcctggat ctgcggtggt2701 caggtttgcc tctgcttttg ttccctgcct ttccgccaca tcacacggcc tcactgggct2761 tcctaccagt ttctcagaat tacacgcacg accatccaaa cattggctcc atgccagggc2821 tgtgactgca gcctgtgtag gaccatgggg agttcagagt catcatacaa ggcagccaag2881 aactgccata aacactacac agatggaggg catgggatag gacaggagag gaggtgatgt2941 acacttagag gtaactaagg ctagtttgaa aaccgtgaat taagatattc ttatcagtat3001 aaaaacactg agattgctta cctcccaaat tgctttattg tattaactct catgcttcag3061 ctcttggatt tgttgtttct aactaacatg tcatgtacac caaacatttt ataaatcaat3121 cagtaaatat tttgtaagac ccaaaatcct ggcatattgc caggtctgtg gatttgcctg3181 ttgggtgttg ggatgggata tcagaggaag aagcatacaa gtgggaaaaa ctgtgattag3241 aaagggaaaa ataggcattt ccacagtact attaagagag gcatgataat gccatttttt3301 ttcttctgta caactggtca gatttcaata aataatcatt gatggtaaaa aaaaaaaaaa3361 aaaa人特异性蛋白激酶-15基因cDNA序列MKEAGQMQNLESARAGRSVSTQTGSMTGQIPRLSKVNLFTLLSLWMELFPAEAQRQKS QKNEEGKHGPLGDNEERTRVSTDKRQVKRTGLVVVKNMKIVGLHCSSEDLHAGQIALIKHGSRLKNCDLYFSRKPCSACLKMIVNAGVNRISYWPADPEISLLTEASSSEDAKLDAKAVERLKSNSRAHVCVLLQPLVCYMVQFVEETSYKCDFIQKITKTLPDANTDFYYECKQERIKEYEMLFLVSNEEMHKQILMTIGLENLCENPYFSNLRQNMKDLILLLATVAS SVPNFKHFGFYRSNPEQINEIHNQSLPQEIARHCMVQARLLAYRTEDHKTGVGAVIWAEGKSRSCDGTGAMYFVGCGYNAFPVGSEYADFPHMDDKQKDREIRKFRYIIHAEQNALTFRCQEIKPEERSMIFVTKCPCDECVPLIKGAGIKQIYAGDVDVGKKKADISYMRFGELEGVSKFTWQLNPSGAYGLEQNEPERRENGVLRPVPQKEEQHQDKKLRLGIH

Amino acid sequence of open reading frame of human specific protein kinase-15 gene

The purpose of the present invention is: in view of the fact that the human specific protein kinase-15 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 a gene related to the specific function of the testis, and its expression is low in embryos and high in adults, it is likely to be related to spermatogenesis in the testis, and its abnormal expression may affect male reproductive function. Therefore, the study of this gene It can be used for: ①Preparing the fusion protein of the gene to study the relationship between the gene and the function of the testis, and may also become a biological drug for treating diseases related to the testis of men.

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 human-specific protein kinase-15 genes can be used for immunohistochemistry Staining, ELISA, immunoelectron microscopy, immunoprecipitation and other detection methods designed according to immunological principles can be used for the localization and quantitative research of the protein encoded by the gene in tissue cells, and can also be used for various biological samples (such as blood, semen, etc.) , urine, etc.) and protein-protein interaction studies. Therefore, the antibody may be used to prepare a diagnostic kit for diseases related to abnormal expression of human specific protein kinase-15 gene, and may be used to prepare 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 Specific Protein Kinase-15 Gene Fusion Protein Prepare GST-SP2-pDEST15 expression plasmid with the nucleotide sequence of the open reading frame of human specific protein kinase-15 gene and plasmid pDEST ^TM 15, in NaCl-free LB Cultured at 30°C to 0.5 OD600, induced by 0.3M NaCl, ultrasonically pulverized and purified by GST affinity chromatography column to obtain purified fusion protein. 2. Human-specific protein kinase-15 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 Adjuvant) 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-specific protein kinase-15 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 Adjuvant) 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 multiple times until a monoclonal hybridoma cell line secreting anti-human specific protein kinase-15 antibody was obtained. 4. Human-specific protein kinase-15 gene is used to prepare testis functional gene expression chip

(1) Design primers based on the obtained nucleotide sequence of the human specific protein kinase-15 gene, amplify the full-length human specific protein kinase-15 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 the gene is used for the diagnosis and drug screening of human-specific protein kinase-15 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 analysis of the intensity of the hybridization signal, the protein expression level of the human specific protein kinase-15 gene or the deletion of the human specific protein kinase-15 gene are affected.

Claims (5)

1. PECIFIC PROTEIN KINASE15, NYD-SP15's gene (NYD-SP15) is cloned the fusion of preparation; It is characterized in that the whole ORFs amino acid of NYD-SP15 gene, sequence is: MKEAGQMQNLESARAGRSVSTQTGSMTGQIPRLSKVNLFTLLSLWMELFPAEAQRQ KSQKNEEGKHGPLGDNEERTRVSTDKRQVKRTGLVVVKNMKIVGLHCSSEDLHAGQ IALIKHGSRLKNCDLYFSRKPCSACLKMIVNAGVNRISYWPADPEISLLTEASSSE DAKLDAKAVERLKSNSRAHVCVLLQPLVCYMVQFVEETSYKCDFIQKITKTLPDAN TDFYYECKQERIKEYEMLFLVSNEEMHKQILMTIGLENLCENPYFSNLRQNMKDLI LLLATVASSVPNFKHFGFYRSNPEQINEIHNQSLPQEIARHCMVQARLLAYRTEDH KTGVGAVIWAEGKSRSCDGTGAMYFVGCGYNAFPVGSEYADFPHMDDKQKDREIRK FRYIIHAEQNALTFRCQEIKPEERSMIFVTKCPCDECVPLIKGAGIKQIYAGDVDV GKKKADISYMRFGELEGVSKFTWQLNPSGAYGLEQNEPERRENGVLRPVPQKEEQH QDKKLRLGIH

2. according to the described polyclonal antibody at the preparation of NYD-SP15 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-SP15 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-SP15 gene cDNA according to claim 1 is described at NYD-SP15 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-SP15 gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the medicine of preparation treatment relative disease.