CN1318562A - Human testicular development specific protein-14 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 development specific protein gene-14 (testis development specific protein gene, NYD-SP14) gene is 1760bp, its open reading frame sequence is 1401bp, and it encodes 466 Amino acid, the number of this gene in Genbank (Genbank) is AF349510. The present invention utilizes NYD-SP14 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 for the treatment of related diseases. gene medicines.

Description

Human testis development specific protein-14 gene encoding protein

The invention belongs to the technical field of human genome.

The human testis development specific protein-14 gene (testis development specific proteingene, NYD-SP14) 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 firstly 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 by sequence determination to be full-length cDNA, which was 1760bp; its open reading frame was 1401bp, which encoded 466 amino acids. Check the gene bank, the gene is new. Since this gene is highly expressed in adult testis, the human testis development-specific protein-14 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 a testis functional gene expression chip. Fusion proteins may also become gene medicines for the treatment of related diseases.

Since there is no homologous gene of human testis development specific protein-14 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 BiotechnologyInformation,NCBI)已正式接受该基因为Genbank的新成员，接受号为AF349510。1 gggggagcca ggcttcggaa tcactgagag aggagggggc agcgccaacg cccagcacct61 gccgccggtg ggggactgac ttcggcggaa cggaaaactg gagttgtttc cctgggccag121 tgcaccgagg ctgaagtcta taagcctgga gagaccatca gcagtaatct gcaaattccg181 aaatgaccac cctgccccca ctgcccatga cacgcccgaa gcttacagcc ttagccagac241 agaagctgcc ttgctcctcc agaaaaattc caaggtctca attgatcaaa gaaaaagatg301 acatagatca ttatctagag gtaaatttca aaggattatc aaaagaggaa gttgctgctt361 ataggaactc ctacaagaag aatatctgtg tggacatgct gcgagatggt tatcataagt421 ccttcaccga gctcttcgct ctgatggagc ggtgggatgc cctgagggag gctgcgagag481 tcaggtccct cttctggctg cagaagcccc tggaggagca gcctgataaa ctggattacc541 tgtaccatta cctgaccagg gctgaggacg ctgagaggaa agaatccttc gaagatgtat601 ataataactt gtatgctctg gcctgttact tcaataattc tgaagacaag tgggtaagga661 accacttcta tgaacgatgt tttaagattg ctcagctgat caaaattgac tgtgggaaga721 aagaagccga ggcacacatg catatgggtc ttctctacga ggaagatggt cagcttctgg781 aagctgctga gcattatgaa gcattccatc aattgacaca ggggcggata tggaaggatg841 agacaggccg ctctctcaac ttgttggcct gtgagagtct cctgaggact tacagattac901 tctcagacaa aatgctagaa aataaagaat acaaacaggc catcaaaatt ctaataaaag961 cttctgaaat agccaaagaa ggaagtgaca aaaagatgga agcggaaacc tcttactact1021 tgggcttagc acacttagct gctgaggaat atgaaacagc attaacagtc cttgacactt

1081 actgtaaaat ctccacagac ctggatgatg atctcagtct ggggagaggc tatgaagcca

1141 tagccaaggt cctgcagagc caaggagaga tgacagaagc aattaaatac ttgaaaaaat

1201 ttgtgaaaat tgcaagaaac aattttcaaa gcctagattt ggtgagagca agtacaatgc

1261 ttggggacat ctacaatgaa aaaggatact acaacaaagc ttctgaatgc tttcagcaag

1321 cttttgacac aacagtagag ctaatgagca tgcctctgat ggatgagaca aaagttcact

1381 atggaatagc aaaagctcat cagatgatgc ttacagtgaa caactatata gagtctgcag

1441 atctcaccag cctcaactac ctgctgtcat ggaaggagag cagaggtaac attgaacctg

1501 atccagttac tgaagagttt agaggatcca cagtggaagc tgtatctcaa aactcagaac

1561 gtttggaaga actcagtagg taggtttcca ggtgatcaaa aaaatgaaac ttaaagcagc

1621 ttttcactta acatcaaagc aagaagaaat ccatcatgtc accttagact tcaatacttg

1681 taataggagc atgttacaga taatatagag ttataataaa acggactatg actcttttca

1741 aaaaaaaaaaaaaaaaaaaa

人睾丸发育特异性蛋白-14cDNA序列MTTLPPLPMTRPKLTALARQKLPCSSRKIPRSQLIKEKDDIDHYLEVNFKGLSKEEVAAYRNSYKKNICVDMLRDGYHKSFTELFALMERWDALREAARVRSLFWLQKPLEEQPDKLDYLYHYLTRAEDAERKESFEDVYNNLYALACYFNNSEDKWVRNHFYERCFKIAQLIKIDCGKKEAEAHMHMGLLYEEDGQLLEAAEHYEAFHQLTQGRIWKDETGRSLNLLACESLLRTYRLLSDKMLENKEYKQAIKILIKASEIAKEGSDKKMEAETSYYLGLAHLAAEEYETALTVLDTYCKISTDLDDDLSLGRGYEAIAKVLQSQGEMTEAIKYLKKFVKIARNNFQSLDLVRASTMLGDIYNEKGYYNKASECFQQAFDTTVELMSMPLMDETKYHYGIAKAHQMMLTVNNYIESADLTSLNYLLSWKESRGNIEPDPVTEEFRGSTYEAVSQNSERLEELSR人睾丸发育特异性蛋白-14开放阅读框架氨基酸序列

The purpose of the present invention is: in view of the fact that the human testis development specific protein-14 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. Because this gene is a gene related to the specific function of testis, and it is low expression in embryo and high expression in adult, so this gene is likely to be related to the development of 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 development-specific protein-14 gene can be used as immune tissue Chemical 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, urine, etc.) content changes and protein-protein interaction research. Therefore, the antibody may be used to prepare a diagnostic kit for diseases related to the abnormal expression of the human testis development-specific protein-14 gene, and may be used to prepare anti-fertility 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 development specific protein-14 gene fusion protein

The nucleotide sequence of the open reading frame of the human testis development specific protein-14 gene and the plasmid pDES ^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 Finally, after ultrasonic pulverization, it was purified by GST affinity chromatography to obtain a purified fusion protein.

2. Human testis development specific protein-14 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 testis development specific protein-14 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 development specific protein-14 antibody was obtained.

4. Human testis development specific protein-14 gene is used to prepare testis function gene expression chip

(1) Design primers based on the obtained nucleotide sequence of the human testis development-specific protein-14 gene to amplify the full-length human testis development-specific protein-14 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 human testis development specific protein-14 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 the human testis development specific protein-14 gene or the deletion of the human testis development specific protein-14 gene are affected.

Claims (5)

1.人睾丸发育特异性蛋白-14基因(NYD-SP14)克隆制备的融合蛋白，其特征是NYD-SP14基因全部开放阅读框氨基酸，序列为：MTTLPPLPMTRPKLTALARQKLPCSSRKIPRSQLIKEKDDIDHYLEVNFKGLSKEEVAAYRNSYKKNICVDMLRDGYHKSFTELFALMERWDALREAARVRSLFWLQKPLEEQPDKLDYLYHYLTRAEDAERKESFEDVYNNLYALACYFNNSEDKWVRNHFYERCFKIAQLIKIDCGKKEAEAHMHMGLLYEEDGQLLEAAEHYEAFHQLTQGRIWKDETGRSLNLLACESLLRTYRLLSDKMLENKEYKQAIKILIKASEIAKEGSDKKMEAETSYYLGLAHLAAEEYETALTVLDTYCKISTDLDDDLSLGRGYEAIAKVLQSQGEMTEAIKYLKKFVKIARNNFQSLDLVRASTMLGDIYNEKGYYNKASECFQQAFDTTVELMSMPLMDETKVHYGIAKAHQMMLTVNNYIESADLTSLNYLLSWKESRGNIEPDPVTEEFRGSTVEAVSQNSERLEELSR 2. According to the polyclonal antibody prepared against the amino acid sequence of the open reading frame of NYD-SP14 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-SP14 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, aiming at using the open reading frame amino acid sequence of NYD-SP14 gene to prepare a testis functional gene expression chip, it is characterized in that NYD-SP14 gene cDNA is amplified by PCR and spotted on the chip for chip preparation. 5. According to claim 1, the fusion protein is prepared according to the amino acid sequence of the open reading frame of the NYD-SP14 gene, which is characterized in that the fusion protein can be used as a medicine for treating related diseases.