CN1309138A - Human testicular cytodifferentiation related protein gene coded protein - Google Patents

Abstract

The invention belongs to the field of human genome technology. The human testicular cell differentiation-related protein gene has a full-length cDNA sequence of 963 bp, an open reading frame of 672 bp, and encodes 224 amino acids. The number of the gene in Genbank (Genbank) is AF288404 . The human testis cell differentiation-related protein gene expression clone of the present invention can be used to prepare a fusion protein, and the protein can be used to immunize animals to prepare monoclonal and polyclonal antibodies, and the gene clone can be used for the preparation of testicular specific function gene expression chips.

Description

Human testis cell differentiation-related protein gene encoding protein

The invention belongs to the technical field of human genome.

The human testis cell differentiation-related protein gene of the present invention was obtained by the inventors in the Key Laboratory of Reproductive Medicine of Nanjing Medical University in Jiangsu Province by using the gene chip method. First, a human testis functional gene expression chip was prepared, and the functional genes of the embryo and adult testis were compared. Differential expression clones were obtained, which were proved to be full-length cDNAs of 963 bp by sequence determination; their open reading frame was 672 bp, and they encoded 224 amino acids. Check the gene bank, the gene is homologous to the mouse pinch protein gene, and the protein expressed by this gene is related to cell differentiation. Since this gene is highly expressed in adult testis, and the main difference between embryo and adult testis is that the latter has spermatogenesis, and spermatogenesis is the process of cell division and differentiation, suggesting that this gene is related to spermatogenesis. 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, and the gene can also be used to prepare a testis function gene expression chip.

Since the human testis cell differentiation-related protein gene has no homologous gene in the Genbank database, the inventors' research results on the structure and function of this gene are all groundbreaking. The National Center for Biotechnology Information (NCBI) has officially accepted the gene as a new member of Genbank, the acceptance number is AF288404.

1 ttctcatccc tgtgctcaga gctctcaagc accacgatg gccttctcag gccgagcgcg

61 cccctgcatt atcccagaga acgaagaaat cccccgagca gcccttaaca ctgtccacga

121 ggccaatggg accgaggacg agagggctgt ttccaaactg cagcgcaggc acagtgacgt

181 gaaagtctac aaggagttct gtgactttta tgcgaaattc aacatggcca acgccctggc

241 cagcgccact tgcgagcgct gcaagggcgg ctttgcgccc gctgagacga tcgtgaacag

301 taatggggag ctgtaccatg agcagtgttt cgtgtgcgct cagtgcttcc agcagttccc

361 agaaggactc ttctatgagg aacgaacgtg atggttaagc taccttcatt aagcagcatg

421 accgcttctg agctcctgct gccgcattta ggccctgtca gctgtgaggc cactggtgct

481 gagtcaaagg gaaaagctca gaacagcctt gacagcgaag aataaagcat gactcatctc

541 gtcctgccca cccctcacac tggactataa ataccctccc tgggtctgtg tgctgttatt

601 ccaaaagatg cgatgaatcc cagaactagg ctattcatgg gctgttgccc agttcaagct

661 ctattgggtc ctgaggaaga ttactatttt ggctacattg tttatcatta aaaccagccc

721 agggatgtgt aaacctgcat ctggagaata agacatttct ttttggttaa atgatattag

781 agaaaaaaaa aactatccaa ggctgctaac aaataatctt cactttcaat tatatccagc

841 aataagttgt tctccagaaa agaaagcttg gaaacttacc cagtttgcct tccatttggg

901 acatgactga tgtgcttcag tctttgtata ataaatgcat cttcgcccct ataaaaaaaa

961 aaa

Human testicular cell differentiation-related protein gene cDNA sequence MAFSGRARPCIIPENEEIPRAALNTVHEANGTEDERAVSKLQRRHSDVKVYKEFCDFYAKFNMANALASATCERCKGGFAPAETIVNSNGELYHEQCFVCAQCFQQFPEGLFYEERT Human testicular cell differentiation-related protein gene open reading frame amino acid sequence

The purpose of the present invention is: in view of the fact that the human testis cell differentiation-related protein 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 testicular cell differentiation-related proteins 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 the abnormal expression of human testicular cell differentiation-related protein genes, 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 solution of the present invention 1. Preparation of human testis cell differentiation-related protein gene fusion protein

The nucleotide sequence of the open reading frame of the human testis cell differentiation-related protein 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 cell differentiation-related protein 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 cell differentiation-related protein 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 genes related to differentiation-related proteins of human testis cells was obtained. 4. Human testis cell differentiation-related protein gene is used to prepare testis functional gene expression chip

(1) Design primers with the obtained nucleotide sequence of the human testis cell differentiation-related protein gene to amplify the full-length human testicular cell differentiation-related 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; eHsapiens mRNA for 23KD highly basicprotein; f.ubiqnitin C; g.Phospholipase A2; h.ubiquitin carboxy Ⅰ-ferminal esterase L Ⅰ). Phage DNA and P-blue plasmid were added as negative controls. (3) The chip containing the gene is used for the diagnosis and drug screening of human testis cell differentiation-related protein 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 strength of the hybridization signal, it affects the protein expression level of the human testicular cell differentiation-related protein gene protein or the deletion of the human testicular cell differentiation-related protein gene.

Claims (4)

1. A fusion protein prepared by cloning a sperm function-specific gene, which is characterized by amino acids in all open reading frames of human testicular cell differentiation-related protein genes, and its sequence is: MAFSGRARPCIIPENEEIPRAALNTVHEANGTEDERAVSKLQRRHSDVKVYKEFCDFYAKFNMANALASATCERCKGGFAPAETIVNSNGELYHEQCFVCAQCFQQFPEGLFYEERT 2. The polyclonal antibody prepared against the open reading frame amino acid sequence of human testicular cell differentiation-related protein gene according to claim 1 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 claim 1, the monoclonal antibody prepared against the open reading frame amino acid sequence of human testicular cell differentiation-related protein gene is characterized in that the fusion protein is mixed with complete and incomplete adjuvants, immunized with Balb/C mice, and isolated spleen B lymphocytes The cells are fused with myeloma cells to produce hybridoma cells, and the monoclonal secretion of IgG antibodies is further screened by subcloning methods, ELISA and antibody typing methods to prepare monoclonal antibodies. 4. According to claim 1, the amino acid sequence of the open reading frame of the human testicular cell differentiation-related protein gene is used to prepare a testis functional gene expression chip, which is characterized in that PCR amplification of the human testicular cell differentiation-related protein gene cDNA is applied to the chip for the chip preparation.