CN1316435A - Human testicular development specificity protein-8 gene coding protein - Google Patents

Abstract

A human testicular development-spedific protein-8 gene has a full cDNA sequence legnth of 1123 bp, open reading frame sequence of 750 bp, 249 coded amino acids and No.Ay014258 in genbank. Its gene clone can be used to prepare fusion protein and the testicular specific function gene expression chip. Said fusion protein can be used to immunize animal to prepare monoclone and polyclone antibodies. Said expression protein can be used to prepare relative gene medicines.

Description

The human testicular development specificity protein-8 gene proteins encoded

The invention belongs to the Human genome technical group field.

Human testicular development specificity protein-8 gene of the present invention (testis specificprotein 8, NYD-SP8) be that the contriver obtains with the method for gene chip screening in reproductive medicine Jiangsu Province key lab of Nanjing Medical University, at first prepare people's testicular function gene expression chip, by comparing embryo and grownup's testicular function expression of gene, acquisition differential expression clone, prove full-length cDNA through sequencing, be 1123bp; Its open reading frame is 750bp, its 249 amino acid of encoding.Consult gene pool, this gene and mouse testis specific gene TSP101RP homology, therefore the human testicular development specificity protein-8 gene of finding may be similar to mouse TSP101RP.Can be prepared into fusion rotein with this gene clone, with this protein immunization rabbit, can obtain mono-clonal and polyclonal antibody, this gene also can be used for preparing the testicular function gene expression chip.Fusion rotein also may become the genomic medicine for the treatment of correlative diseases

Because human testicular development specificity protein-8 gene still unmanned homologous gene in the Genbank database; the inventor all is initiative for the 26S Proteasome Structure and Function achievement in research of this gene. ( National CenterforBiotechnologyInformation NCBI ) formally accepts the newcomer that this gene is Genbank, and accepting number is AY014258 in American National biotechnology information center.1 gggaggcgtt ccgggcctca actttggcgt cgtgagattc ttgtgaggcg tctgcctgga 61 agccggcagc aattttgctt ctttaaagag aaaaagaagg ctagggactc agattcctgg 121 attctgagat ccagaccagc tcctcccaga cctctccaga agaagccatg ggaacccctc 181 gtatccagca tttgctgatc ctcctggtcc taggagcctc cctcctgacc tcgggcctag 241 agctgtattg tcaaaagggt ctgtccatga ctgtggaagc agatccagcc aatatgttta 301 actggaccac agaggaagtg gagacttgtg acaaaggggc actttgccag gaaaccatac 361 taataattaa agcagggact gagacagcca ttttggccac gaagggctgc atcccggaag 421 gggaggaggc cataacaatt gtccagcact cttcacctcc cggcctgatc gtgacctcct 481 acagtaacta ctgtgaggat tccttctgta atgacaaaga cagcctgtct cagttttggg 541 agttcagtga gaccacagct tccactgtgt caacaaccct ccattgtcca acctgtgtgg 601 ctttggggac ctgtttcagt gctccttctc ttccctgtcc caatggtaca actcgatgct 661 atcaaggaaa acttgagatc actggaggtg gcattgagtc gtctgtggag gtcaaaggct 721 gtacagccat gattggctgc aggctgatgt ctggaatctt agcagtagga cccatgtttg 781 tgagggaagc gtgcccacat cagctgctca ctcaacctcg aaagactgaa aatggggcca 841 cctgtcttcc cattcctgtt tgggggttac agctactgct gccattgctg ctgccatcat 901 ttattcactt ttcctaagaa ggcacttctg ggcctgggtc tgaggacatc ttttttgact 961 gggagccttc ttactgttga ggttcaacaa gctgaggagt agatgggaat ttgagggaga1021 atacagagat actatgaacg tatttgacat ttttaataca atttctgcta taatttttgt1081 atgcagtagg cgttactaat aaacatttct gctaaaaaaa aaa-8cDNAMGTPRIQHLLILLVLGASLLTSGLELYCQKGLSMTVEADPANMFNWTTEEVETCDKGALCQETILIIKAGTETAILATKGCIPEGEEAITIVQHSSPPGLIVTSYSNYCEDSFCNDKDSLSQFWEFSETTASTVSTTLHCPTCVALGTCFSAPSLPCPNGTTRCYQGKLEITGGGIESSVEVKGCTAMIGCRLMSGILAVGPMFVREACPHQLLTQPRKTENGATCLPIPVWGLQLLLPLLLPSFIHFS

Human testicular development specificity protein-8 gene open reading frame aminoacid sequence

The objective of the invention is: in view of human testicular development specificity protein-8 gene is the brand-new gene that the inventor finds, human at present understanding and the understanding to this gene only limits to the scientific research that the inventor does.Because this gene is the gene relevant with the testis specific function, and be the low expression of embryo, adult's high expression level, so this gene is relevant with testicualr development probably, its unconventionality expression may influence the male sex's reproductive function, so the research of this gene can be used for:

1. by this gene Fusion albumen of preparation, be used to study the relation of this gene and testicular function, also may become a kind of biologics for the treatment of the male testical relative disease.

2. prepare specific antibody: the specific antibody of gene coded protein is the indispensable important tool of research gene structure and function, the polyclone of human testicular development specificity protein-8 gene proteins encoded and monoclonal antibody, can be used as immunohistochemical staining, ELISA, immuno-electron microscope, multiple detection method such as co-immunoprecipitation according to the immunology principle design, can be used for location and the quantitative examination of this gene coded protein in histocyte, also can be used for various biological specimens (as blood, seminal fluid, urine etc.) content research in and protein-protein interaction research etc.Therefore, this antibody might be used for preparation and human testicular development specificity protein-8 gene abnormal expression relative disease diagnostic kit, and might be used to prepare antifertility drug.

3. prepare gene diagnosis chip: functional gene is used to prepare the importance that gene diagnosis chip is the gene development research, all significant at aspects such as medical diagnosis on disease (as male sterility, sexual disorder) and drug screenings.The preparation of technical solution of the present invention 1. human testicular development specificity protein-8 gene fusion roteins is with the nucleotide sequence and the plasmid pDEST of human testicular development specificity protein-8 gene open reading frame ^TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, and 30 cultivate 0.5 OD600 in the LB of no NaCl, and after 0.3M NaCl induced, ultrasonication obtained the fusion rotein of purifying after GST affinity chromatography column purification.2. human testicular development specificity protein-8 gene coded protein specific polyclonal antibody

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), through the dialysis after again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) balanced mix, immunity new zealand rabbit (back subcutaneous injection), every 3～4 all booster injections once, totally three times, after three months, get blood system from serum.Be ELISA in contrast with animal serum before the immunity, measure antibody titers in each immunize rabbit serum.Screen high titre serum, further do competition and suppress the specificity that immunity test is determined antibody with antigen.Determine that according to ELISA result its dilution that is used for immunohistochemical staining multiplication number is 1: 200～500.3. human testicular development specificity protein-8 gene coded protein specific monoclonal antibody

Getting fusion rotein combines with carrier proteins KLH (Keyhole Limpet Hymocyanin), after dialysis again with fully/Freund (Complete/Incomplete Freund ' sAdjuvant) mixes, immunity Balb/C mouse (intraperitoneal injection), every other week once, continuous 2-3 time, vein is strengthened once before merging, after ELISA method confirmation production of antibodies, get the spleen separating Morr. cell, merge the generation hybridoma with the myeloma cell then.Positive colony is through subclone repeatedly, until the monoclonal hybridoma strain that obtains the anti-human testicular development specificity protein-8 antibody of secretion.4. human testicular development specificity protein-8 gene is used to prepare the testicular function gene expression chip

(1) with the nucleotide sequence design primer of the human testicular development specificity protein-8 gene that obtains, the human testicular development specificity protein-8 gene sequence of amplification total length is as the sample of some film.(2) with Britain BioRobotics put automatically the film instrument with specimens point on 8 * 12cm nylon membrane, 2 points of each specimens point, several approximately ng of every DNA amount. 8 housekeeping genes of positive control (a.ribosomal protein S9; B.Actin gamma; C.glyceraldehyde-3-phosphate dehydrogenase; D.hypoxanthinephosphoribosyltransferase 1; E.H.sapiens mRNA for 23KD highly basicprotein; F.ubiqnitin C; G.Phospholipase A2; H.ubiquitin carboxy I-ferminal esterase L I).Negative control adds lambda bacteriophage dna and P-blue plasmid.(3) chip that will contain this gene is used for the diagnosis and the drug screening of human testicular development specificity protein-8 gene disappearance ³³The P mark:

Extracting sample mRNA to be checked or DNA are with Random Primer labelling method mark sample mRNA to be checked or DNA, as the probe of hybridization.Hybridization:

After the nylon membrane label probe hybridization that point is made, oven dry is shielded compressing tablet with phosphorus in the hybrid heater (68 ℃).Signal scanning and analysis:

Analyze hybridization signal with registering instrument.According to the strong and weak analyzing influence human testicular development specificity protein-8 gene protein expression level of hybridization signal or the disappearance of human testicular development specificity protein-8 gene.

Claims (5)

1. human testicular development specificity protein-8 (NYD-SP8) is cloned the fusion rotein of preparation, it is characterized in that the whole open reading frame amino acid of NYD-SP8 gene, sequence is: MGTPRIQHLLILLVLGASLLTSGLELYCQKGLSMTVEADPANMFNWTTEEVETCDK GALCQETILIIKAGTETAILATKGCIPEGEEAITIVQHSSPPGLIVTSYSNYCEDS FCNDKDSLSQFWEFSETTASTVSTTLHCPTCVALGTCFSAPSLPCPNGTTRCYQGK LEITGGGIESSVEVKGCTAMIGCRLMSGILAVGPMFVREACPHQLLTQPRKTENGA TCLPIPVWGLQLLLPLLLPSFIHFS

2. according to the described polyclonal antibody at the preparation of NYD-SP8 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-SP8 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-SP8 gene cDNA according to claim 1 is described at NYD-SP8 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-SP8 gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the medicine of preparation treatment relative disease.