CN1309137A - Human testicular gastrulation protein gene coded protein - Google Patents

Abstract

The invention belongs to the field of human genome technology. The human testis gastrulation protein gene (twisted gastrulation protein) has a full-length cDNA sequence of 1949bp, its open reading frame is 672bp, and encodes 224 amino acids. The gene is listed in Genbank (Genbank) The number in it is AF294628. The human testis gastrulation 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 testis-specific function gene expression chips.

Description

human testis gastrulation protein gene encoding protein

The invention belongs to the technical field of human genome.

The human testicular gastrulation protein gene 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. First, a human testis functional gene expression chip was prepared, and by comparing embryonic and adult testicular functional genes Differential expression clones were obtained, which were proved to be full-length cDNA of 1949 bp by sequence determination; its open reading frame was 672 bp, and it encoded 224 amino acids. Consulting the gene bank, this gene is homologous to the gastrulation protein gene of mouse and Xenopus laevis, and the main function of the protein expressed by this gene is to activate bone morphogenic protein. Since this gene is highly expressed in adult testes, the main difference between embryos and adult testes is that the latter has spermatogenesis. At the same time, our laboratory used in situ hybridization to show that this gene is low expressed in spermatogonia, but not in spermatogonia. The high expression of the later spermatogenic cells suggested that the gene may be the initiator of 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 testicular gastrulation 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 AF294628.

1 tctttgaaga aacatgaagt tacactatgt tgctgtgctt actctagcca tcctgatgtt

61 cctgacatgg cttccagaat cactgagctg taacaaagca ctctgtgcta gtgatgtgag

121 caaatgcctc attcaggagc tctgccagtg ccggccggga gaaggcaatt gctcctgctg

181 taaggagtgc atgctgtgtc ttggggccct ttgggacgag tgctgtgact gtgttggtat

241 gtgtaatcct cgaaattata gtgacacacc tccaacttca aagagcacag tggaggagct

301 gcatgaaccg attccttctc tcttccgggc actcacagaa ggagatactc agttgaattg

361 gaacatcgtt tctttccctg ttgcagaaga actttcacat catgagaatc tggtttcatt

421 tttagaaact gtgaaccagc cacaccacca gaatgtgtct gtccccagca ataatgttca

481 cgcgccttat tccagtgaca aagaacacat gtgtactgtg gtttatttg atgactgcat

541 gtccatacat cagtgtaaaa tatcctgtga gtccatggga gcatccaaat atcgctggtt

601 tcataatgcc tgctgcgagt gcattggtcc agaatgtatt gactatggta gtaaaactgt

661 caaatgtatg aactgcatgt tttaaagaag acaaatgcaa accaaagcaa cttagtaaaa

721 taataggtat aaaaagttat tctgtaagtc tgttggttgt atcttgtatc aaaatcccag

781 taagttaagt tgtaaagact ttggaataag tttcttttta aaaatatgac attagccagg

841 gatgtgttta atttataac tggtcttact gattttatg ccccctagca ataagccctt

901 tcctttgatt catgtacaaa tttggtcata tgagaagcag gtgcgcagag aattccttga

961 aagatatgag gtttttaaca tgaagtctga tgtggttttc ctctagcatt ccaaaaegtt

1021 tttgctttga aagtgttagc agaagcatgt tgatgtgaat tatgatttct tcatgtgcta

1081 ctgttagcac actgagtttt tatagttgca catcattcct cattgtgcct tgttttatcc

1141 attttataaa tagagtagat atttgatata ccactctgat aactcatata aaaatatcat

1201 cataaaaagc ttaatttcat cccttttatg ttggttttaa aaggtaaatg cttaccatat

1261 tttataattg agaactctta catagtagaa tccattctat aatacatgtg ttgacaaagc

1321 tttagagaaa gtttcctatt ccccctgccc aaaaggcttg acanaggcag tgatgaaaaa

1381 tcttttacca agattttcag ggtgtaccta tgaaattgct ttaaatgcac tgctggtgta

1441 aataattagc aagcaaatgc gtttctgtga cttcaggtac cagcttaaag agcactaggg

1501 atggggaacg aatgccaaat cagactccac ctagagcacc aggaaacagc ttgtaccctg

1561 gtagggaaat ggtgttgctg aaaggggagg ctgagccagt gcgagactga acttgtgcag

1621 ccttagccaa gacaaagcag tgtttttcag cagacggctg atgggacagg aattgaagaa

1681 gagaattgac tcgtatgaac aggacagggt gaaaatgctg ggaattataa tgggaaacaa

1741 aactatctat gttcatattt tgtaatattt catttgttaa gtttatatct ggatataatg

1801 ttctttttaa acaagtataa tcatatcgtc ggaggttaag attatgaaat tttagaatct

1861 ctattcaaga tgatgttcac tccaaataca ctacagaatt tagtcaacat tttatataat

1921 gtttcaataa atgtttcttt ccccaaaaa

人睾丸原肠形成蛋白基因cDNA序列MKLHYVAVLTLAILMFLTWLPESLSCNKALCASDVSKCLIQELCQCRPGEGNCSCCKECMLCLGALWDECCDCVGMCNPRNYSDTPPTSKSTVEELHEPIPSLFRALTEGDTQLNWNIVSFPVAEEISHHENLVSFLETVNQPHHQNVSVPSNNVHAPYSSDKEHMCTVVYFDDCMSIHQCKISCESMGASKYRWFHNACCECIGPECIDYGSKTVKC

Amino acid sequence of open reading frame of human testis gastrulation protein gene

The purpose of the present invention is: in view of the fact that the human testis gastrulation 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 human testis gastrulation protein gene-encoded 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 the human testis gastrulation protein 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 fusion protein of human testis gastrulation protein gene The nucleotide sequence of the open reading frame of human testis gastrulation protein gene and plasmid pDEST ^TM 15 was used to prepare the GST-SP2-pDEST15 expression plasmid, and the expression plasmid was prepared in NaCl-free LB at 30°C Cultured 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 testicular gastrulation 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 testicular gastrulation 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 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 the gene of anti-human testicular gastrulation protein was obtained. 4. Human testis gastrulation protein gene was used to prepare testis functional gene expression chip

(1) Design primers with the obtained nucleotide sequence of the human testis gastrulation protein gene, amplify the full-length human testis gastrulation 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 carboxyⅠ-ferminal esterase LI). 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 gastrulation 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 strength analysis of the hybridization signal, the protein expression level of the human testis gastrulation protein or the deletion of the human testis gastrulation protein gene is affected.

Claims (4)

1. a sperm function specific gene is cloned the fusion rotein for preparing, it is characterized in that people's testis primitive gut forms the whole open reading frame amino acid of protein gene, sequence is: MKLHYVAVLTLAILMFLTWLPESLSCNKALCASDVSKCLIQELCQCRPGEGNCSCC KECMLCLGALWDECCDCVGMCNPRNYSDTPPTSKSTVEELHEPIPSLFRALTEGDT QLNWNIVSFPVAEELSHHENLVSFLETVNQPHHQNVSVPSNNVHAPYSSDKEHMCT VVYFDDCMSIHQCKISCESMGASKYRWFHNACCECIGPECIDYGSKTVKC

2. form the polyclonal antibody of protein gene open reading frame aminoacid sequence preparation according to claim 1 is described at people's testis primitive gut, 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 that forms the preparation of protein gene open reading frame aminoacid sequence at people's testis primitive gut of claim l, 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. be used to prepare the testicular function gene expression chip according to claim 1 is described at people's testis primitive gut being formed protein gene open reading frame aminoacid sequence, it is characterized in that pcr amplification people testis primitive gut forms protein gene cDNA, put in chip and be used for the chip preparation.