CN1313342A - Human testis lactate dehydrogenase A gene coded protein - Google Patents

Abstract

The invention belongs to the field of human genome technology. The full-length cDNA sequence of the human testis novel lactate dehydrogenase A gene (Novel lactate dehydrogenase A) gene is 1680bp, its open reading frame is 1146bp, and encodes 382 amino acids. (Genbank) number AY009108. Using the novel human testis lactate dehydrogenase A gene expression clone of the present invention, a fusion protein can be prepared, the protein can be used to immunize animals to prepare monoclonal and polyclonal antibodies, and the gene cloning can be used for the preparation of testis-specific functional gene expression chips .

Description

Human testis novel lactate dehydrogenase A gene encoding protein

The invention belongs to the technical field of human genome.

The novel human testis lactate dehydrogenase A gene described in the present invention was obtained by the inventors in Jiangsu Key Laboratory of Reproductive Medicine of Nanjing Medical University by gene chip method. For the expression of functional genes, differentially expressed clones were obtained, which were proved to be full-length cDNAs of 1680 bp by sequence determination: its open reading frame was 1146 bp, and it encoded 382 amino acids. Checking the gene bank, it can be seen that there is a homologous gene in the mouse body, which is the lactate dehydrogenase A gene, but it has no homology with the human lactate dehydrogenase gene. Since this gene is highly expressed in adult testes, and the main difference between embryos and adult testes is that the latter has spermatogenesis, the new lactate dehydrogenase A discovered in human testis is likely to be related to spermatogenesis. Cloning with this gene can be prepared into The fusion protein can be used to immunize rabbits to obtain monoclonal and polyclonal antibodies, and the gene can also be used to prepare a testis functional gene expression chip.

Since the novel lactate dehydrogenase A gene of human testis 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 BiotechnologyInformation,NCBI)已正式接受该基因为Genbank的新成员，接受号为AY009108。1 agagtgctga aggtcctgcc aacggctctc ttggcgtctc aacgttcgga tcagcagctt61 ttttccattc tctctctcca cttcttcagt gagcagccat gagttggact gtgcctgttg121 tgcgggccag ccagagagtg agctcggtgg gagcgaattt cctatgcctg gggatggccc181 tgtgtccgcg tcaagcaacg cgcatcccgc tcaacggcac ctggctcttc acccccgtga241 gcaagatggc gactgtgaag agtgagctta ttgagcgttt cacttccgag aagcccgttc301 atcacagtaa ggtctccatc ataggaactg gatcggtggg catggcctgc gctatcagca361 tcttattaaa aggcttgagt gatgaacttg cccttgtgga tcttgatgaa gacaaactga421 agggtgagac gatggatctt caacatggca gccctttcac gaaaatgcca aatattgttt481 gtagcaaaga ttactttgtc acagcaaact ccaacctagt gattatcaca gcaggtgcac541 gccaagaaaa gggagaaacg cgccttaatt tagtccagcg aaatgtggcc atcttcaagt601 taatgatttc cagtattgtc cagtacagcc cccactgcaa actgattatt gtttccaatc661 cagtggatat cttaacttat gtagcttgga agttgagtgc atttcccaaa aaccgtatta721 ttggaagcgg ctgtaatctg gatactgctc gttttcgttt cttgattgga caaaagcttg781 gtatccattc tgaaagctgc catggatgga tcctcggaga gcatggagac tcaagtgttc841 ctgtgtggag tggagtgaac atagctggtg tccctttgaa ggatctgaac tctgatatag901 gaactgataa agatcctgag caatggaaaa atgtccacaa agaagtgact gcaactgcct961 atgagattat taaaatgaaa ggttatactt cttgggccat tggcctatct gtggccgatt1021 taacagaaag tattttgaag aatcttagga gaatacatcc agtttccacc ataattaagg1081 gcctctatgg aatagatgaa gaagtattcc tcagtattcc ttgtatcctg ggagagaacg1141 gtattaccaa ccttataaag ataaagctga cccctgaaga agaggcccat ctgaaaaaaa1201 gtgcaaaaac actctgggaa attcagaata agcttaagct ttaaagttgc ctaaaactac1261 cattccgaaa ttattgaaga gatcatagat acaggattat ataacgaaat tttgaataaa1321 cttgaattcc taaaagatgg aaacaggaaa gtaggtagag tgattttcct atttatttag1381 tcctccagct cttttattga gcatccacgt gctggacgat acttatttac aattcctaag1441 tatttttggt acctctgatg tagcagcact tgccatgtta tatatatgta gttggcattt1501 ggttcccaaa aagtaggatg taggtattta ttgtgttcta gaaattccga ctcttttcat1561 tagatatatg ctatttcttt cattcttgct ggtttatacc tatgttcatt tatatgctgt1621 aaaaaagtag tagcttcttc tacaatgtaa aaataaatgt acatacaaaa aaaaaaaaaa人睾丸新型乳酸脱氢酶A基因cDNA序列MSWTVPVVRASQRVSSVGANFLCLGMALCPRQATRIPLNGTWLFTPVSKMATVKSELIERFTSEKPVHHSKVSIIGTGSVGMACAISILLKGLSDELALVDLDEDKLKGETMDLQHGSPFTKMPNIVCSKDYFVTANSNLVIITAGARQEKGETRLNLVQRNVAIFKLMISSIVQYSPHCKLIIVSNPVDILTYVAWKLSAFPKNRIIGSGCNLDTARFRFLIGQKLGIHSESCHGWILGEHGDSSVPVWSGVNIAGVPLKDLNSDIGTDKDPEQWKNVHKEVTATAYEIIKMKGYTSWAIGLSVADLTESILKNLRRIHPVSTIIKGLYGIDEEVFLSIPCILGENGITNLIKIKLTPEEEAHLKKSAKTLWEIQNKLKL人睾丸新型乳酸脱氢酶A基因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 novel lactate dehydrogenase A gene of human testis 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.

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 new lactate dehydrogenase A gene of human testis can be used as immune tissue Chemical staining, ELlSA, 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 change research and protein-protein interaction research, etc. Therefore, the antibody may be used to prepare a diagnostic kit for diseases related to the abnormal expression of the new lactate dehydrogenase A gene in human testis, 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 Novel Lactate Dehydrogenase A Gene Fusion Protein from Human Testis

The GST-SP2-pDEST15 expression plasmid was prepared with the nucleotide sequence of the open reading frame of the new human testis lactate dehydrogenase A gene and the plasmid pDEST ^TM 15, cultured in LB without NaCl at 30C 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 novel lactate dehydrogenase A 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 novel lactate dehydrogenase A 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 a new type of lactate dehydrogenase A resistant to human testis was obtained. 4. A novel human testis lactate dehydrogenase A gene was used to prepare a testis functional gene expression chip

(1) Design primers with the obtained nucleotide sequence of human testis novel lactate dehydrogenase A gene, amplify the full-length human testis novel lactate dehydrogenase A sequence, and use it as a sample for spotting 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-ferminalesterase 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 novel lactate dehydrogenase A deficiency 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 expression level of human testis novel lactate dehydrogenase A protein or the deletion of human testis novel lactate dehydrogenase A gene.

Claims (4)

1. the fusion rotein of sperm function specific gene clone preparation is characterized in that people's testis is new

The whole ORFs amino acid of type lactate dehydrogenase A gene, sequence is: MSWTVPVVRASQRVSSVGANFLCLGMALGPRQATRIPLNGTWLFTPVSKMATVKSE LIERFTSEKPVHHSKVSIIGTGSVGMACAISILLKGLSDELALVDLDEDKLKGETM DLQHGSPFTKMPNIVCSKDYFVTANSNLVIITAGARQEKGETRLNLVQRNVAIFKL MISSIVQYSPHCKLIIVSNPVDILTYVAWKLSAFPKNRIIGSGCNLDTARFRFLIG QKLGIHSESCHGWILGEHGDSSVPVWSGVNIAGVPLKDLNSDIGTDKDPEQWKNVH KEVTATAYEIIKMKGYTSWAIGLSVADLTESILKNLRRIHPVSTIIKGLYGIDEEV FLSIPCILGENGITNLIKIKLTPEEEAHLKKSAKTLWEIQNKLKL

2. according to the described polyclonal antibody at the preparation of human testis lactate dehydrogenase A 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 human testis lactate dehydrogenase A 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. described at human testis lactate dehydrogenase A gene open reading frame aminoacid sequence is used to prepare the testicular function gene expression chip according to claim 1, it is characterized in that pcr amplification human testis lactate dehydrogenase A gene cDNA, put in chip and be used for chip preparation.