CN1318560A - Human testicular specific protein-1 gene encoded protein - Google Patents

Abstract

The present invention belongs to the field of human genome technology. The full-length cDNA sequence of the human testis specific protein 1 (NYD-TSP1) gene is 1848bp, its open reading frame sequence is 1293bp, and encodes 430 amino acids. The accession number in Genbank (Genbank) is AF333098. The invention utilizes NYD-TSP1 gene cloning to prepare fusion protein, and uses the protein to immunize animals to prepare monoclonal and polyclonal antibodies. At the same time, the gene cloning is used for the preparation of testicular specific function gene expression chips, and the expressed protein may also become a gene for treating related diseases. drug.

Description

human testis specific protein-1 gene encoding protein

The invention belongs to the technical field of human genome.

The human testis specific protein-1 gene (Testis specific protein 1, NYD-TSP1) of the present invention was obtained by the inventor in the Key Laboratory of Reproductive Medicine of Jiangsu Province, Nanjing Medical University, through gene chip method screening, firstly, a human testis functional gene expression chip was prepared, 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 1848 bp by sequence determination; its open reading frame was 1293 bp, encoding 430 amino acids. Check the gene bank, the gene is a new gene, and the protein expressed by the gene is a testis-specific protein. Since this gene is highly expressed in adult testis, the human testis-specific protein gene discovered may be related to testis development, and 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 testis-specific protein-1 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 Biotechnology Information,NCBI)已正式接受该基因为Genbank的新成员，接受号为AF333098。1 ggggggagcc ccagccttgg tcccctttcc cacccttcct ccccacaagg ctgtctgaga61 tgccaccttc cacctaaaca tcatctccca aattttaatc cttaactttg gtctctgact121 tctgcttgat tccacagtct ccacccacat ttgcacaaag gaccatcacc tgtccttcct181 ggaatctcta aagttttctg ctttccttct gtcctgatgg ccagctctgc taagtcagct241 gagatgccca ccatctccaa aacccttaac cctactcctg atcctcatca agaatatctg301 gactctagga ttaccattgc cttattcgaa attggatcac attccccttc ctcctggggc361 tctctccctt tcctaaagaa tagcagccat caagttacag aacaacagac tgcacagaag421 tttaacaatc tcttaaaaga aattaaagat attcttaaaa atatggcagg ttttgaagag481 aagatcacag aagcaaaaga actttttgag gaaaccaata ttactgagga tgtgtcagcc541 cacaaagaaa atatcagagg acttgacaaa atcaatgaaa tgttatcaac aaacctgcct601 gttagtttag ccccagagaa agaagacaat gaaaagaaac aggagatgat attggaaacc661 aatattactg aggatgtgtc agcccacaaa gaaaatatca gaggacttga caaaatcaat721 gaaatgttat caacaaacct gcctgttagt ttagccccag agaaagaaga caatgaaaag781 aaacagcaga tgataatgga aaaccagaac tctgagaaca ccgcacaagt ttttgcaaga841 gatttggtaa atcgtttaga agaaaaaaaa gtccttaacg aaactcaaca aagtcaggaa901 aaagcaaaaa acagacttaa tgttcaagaa gaaactatga aaattaggaa caacatggag961 cagttactac aggaagcaga acactggagt aaacaacata ctgagctcag taaactgata1021 aaatcctatc agaaatctca gaaagacata agtgaaactc ttggaaataa tggagtcggt1081 ttccaaaccc agccaaataa tgaagtgtcg gctaagcctg agctggagga acaggtgaag1141 aaactgagcc atgacaccta ttcattgcag ttgatggcag ctttgctaga gaatgaatgc1201 caaatcttac agcagagagt agagattctc aaggaactcc atcatcagaa acagggaact1261 ctgcaagaga agccaattca gataaactat aaacaggaca agaaaaatca gaagccatca1321 gaagcaaaga aagtagaaat gtataagcag aacaagcaag caatgaaggg tacattttgg1381 aaaaaagaca gatcctgtag aagcctggat gtttgtctta ataagaaagc ttgcaatacc1441 cagttcaata ttcatgttgc aagaaaagct cttaggggaa aaatgaggtc agctagcagc1501 ctaagataga aaataccaaa agcagatgaa aaggtgaatc cttaaaaaac tacatctgtt1561 acctccaact tgtcagtcat gatcaatcat caaaccttgg gcagatctgc tggttcaacc1621 aagaaagagc tatagaataa ggagattgac acttatatta ctcattactt cagcagttat1681 gtaagtctgg tctttaatga ccattgtgtc tacatcaagt tcataacttt tcacctttac1741 aatctgcttg ttttttttgt aattattaga tagacagcct agagtgagaa gttaaaggtc1801 caattaagta aagatctaaa taa a aaccaga aaccca a

人睾丸特异蛋白-1基因cDNA序列MASSAKSAEMPTISKTLNPTPDPHQEYLDSRITIALFEIGSHSPSSWGSLPFLKNSSHQVTEQQTAQKFNNLLKEIKDILKNMAGFEEKITEAKELFEETNITEDVSAHKENIRGLDKINEMLSTNLPVSLAPEKEDNEKKQEMILETNITEDVSAHKENIRGLDKINEMLSTNLPVSLAPEKEDNEKKQQMIMENQNSENTAQVFARDLVNRLEEKKVLNETQQSQEKAKNRLNVQEETMKIRNNMEQLLQEAEHWSKQHTELSKLIKSYQKSQKDISETLGNNGVGFQTQPNNEVSAKPELEEQVKKLSHDTYSLQLMAALLENECQILQQRVEILKELHHQKQGTLQEKPIQINYKQDKKNQKPSEAKKVEMYKQNKQAMKGTFWKKDRSCRSLDVCLNKKACNTQFNIHtVARKALRGKMRSASSLR

Amino acid sequence of open reading frame of human testis specific protein-1 gene

The purpose of the present invention is: in view of the fact that the human testis-specific protein-1 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 Can be used as:

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.

②Preparation of specific antibodies: specific antibodies to gene-encoded proteins are an indispensable and important tool for studying gene structure and function. Polyclonal and monoclonal antibodies to proteins encoded by the human testis-specific protein-1 gene can be used for immunohistochemical 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, research on content changes in urine, etc.) and protein-protein interaction studies, etc. Therefore, the antibody may be used to prepare a diagnostic kit for diseases related to abnormal expression of the human testis-specific protein-1 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 scheme of the present invention

1. Preparation of Human Testis Specific Protein-1 Gene Fusion Protein

The nucleotide sequence of the open reading frame of the human testis-specific protein-1 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 0.5OD600, and induced by 0.3M NaCl, After ultrasonic pulverization, it was purified by GST affinity chromatography column to obtain purified fusion protein.

2. Human testis-specific protein-1 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 times for immunohistochemical staining were determined to be 1:200-500.

3. Human testis-specific protein-1 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 testis-specific protein-1 antibody was obtained.

4. Human testis-specific protein gene is used to prepare testis function gene expression chip

(1) Design primers with the obtained nucleotide sequence of the human testis-specific protein gene, amplify the full-length human testis-specific protein gene sequence, and use it as a sample for spotting the membrane.

(2) Use the British BioRobotics automatic film spotting instrument to spot the specimens 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; e. H. sapiens mRNA for 23KD highly basic protein; f. ubiqnitin C; g. Phospho2 ; 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 the deletion of the human testis specific protein gene

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 testis specific protein gene or the deletion of the human testis specific protein gene is affected.

Claims (5)

1. human testis specific protein 1 gene (NYD-TSP1) is cloned the fusion of preparation; NYD-TSP1,：MDNRKEPPFFNDDNMGPFYYRLHFCDTMELFIETLTGTCFELRVSPFETVISVKAKIRRLEGIPICRQHLIWNNMELENDYCLNDYNISEGCTLKLVLAMRGGPINTRRVPTDDPLRKMAEYLDSSRVEVWEKTSCSKQVTFLVYREGDQLNFFPAVDRGDGTLTPLSDSSKKIDFHLHVLRRKGEHRMSGGSMYNSDTDEDEETEPSSSGQQIIENSITMNKMKLLKAKMKNMNLSKKPKKAVKIKPHPPVAPRPSSGSTAPSRHRLLRVLPNIGQSCSPAFGNAYPPEISRNGISSLATQLSAERYISSITGEFLKEDNSWENNTLSHFSSNVKLPPQIPHLELGNDQELADSVLHLGSSLPRQTKHFLGNLPSSNGNIVLPSEECVTEQSLLPKVGSPASFAEGNADEQSSGLEGACKVNLELLLTNADKGLKAPEQHLKHVAGVLNGESVETSVLNYRELSPHKNRLLSPLRCSASMSLHNSLVKPERQSKCFEFGKLQPSSSQSLDVQNITDSSFSRTTCFQGVKVDSLGKRSDVISKVEARDITEMTNKASKEPVGCVNNISFLASLAGSTSRNRLQSTRGAGRLQNSGTGLSTNLQHFQEENFRKSSPQLEHTGVFLSTHGVGMNGNNAAAGKSVGECTTHHLPPVKAPLQTKKKTTNHCFLCGKKTGLASSYECRCGNNFCASHRYAETHGCTYDYKSAGRRYLHEANPVVNAPKLPKI

2. according to the described polyclonal antibody at the preparation of NYD-TSP1 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-TSP1 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-TSP1 gene cDNA according to claim 1 is described at NYD-TSP1 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-TSP1 gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the genomic medicine of treatment relative disease.