CN1318559A - Human sperm skelemin gene encoded protein - Google Patents
Human sperm skelemin gene encoded protein Download PDFInfo
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- CN1318559A CN1318559A CN 01113507 CN01113507A CN1318559A CN 1318559 A CN1318559 A CN 1318559A CN 01113507 CN01113507 CN 01113507 CN 01113507 A CN01113507 A CN 01113507A CN 1318559 A CN1318559 A CN 1318559A
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Abstract
The present invention belongs to the field of human genome technology. The human sperm skelemin (CALICIN) gene has whole length cDNA sequence of 1943 bp, open reading frame sequence 1767 bp, 588 encoded amino acids and Genebank number of AF333334. The present invention utilizes CALICIN gene clone to prepare fusion protein, utilizes the protein to immunize animal and to prepare monoclonal and polyclonal antibody, and utilizes gene clone in preparing testicular specific function gene expressing chip. The expressed protein may become gene medicine for treating related disease.
Description
The invention belongs to the Human genome technical group field.
Human sperm skelemin gene of the present invention (CALICIN) is 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 total length eDNA through sequencing, be 1943bp; Its open reading frame is 1767bp, its 588 amino acid of encoding.Consult gene pool, this gene and mouse calicin dna homolog, this genetic expression albumen are the specific skelemin of sperm head.Because this gene is the adult testis high expression level, and the main difference of embryo and adult testis is the latter spermatogeny is arranged, therefore the human sperm skelemin gene of finding is relevant with the formation of skelemin in the spermatogeny process probably, 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 of treatment relative disease.
Because human sperm skelemin 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. American National biotechnology (Naional Center for BiotechnologyInformation of information center; NCBI ) Genbank,AF333334. 1 tctccctggt caaccgctct gcaaacaacc atcaatctga tcccacaggc ctgagaaagt 61 ctgctctcca gtacctgctg ctgatctgtt tcagccgaca agaggcacca tgaaattgga 121 attcacggag aaaaactaca atagcttcgt gctgcagaac ctgaacagac agaggaaacg 181 caaagagtac tgggacatgg ccctgagcgt ggacaaccac gtcttctttg cacatcgcaa 241 tgtgctggct gctgtctccc cactggtgag gagcctcatc tccagcaatg acatgaagac 301 cgctgatgag cttttcatca ccattgacac cagttacctg agcccggtca cagtggacca 361 gcttctggac tacttctata gcggcaaggt ggtgatctcc gagcaaaatg tggaggagct 421 gcttcgtggg gctcagtatt tcaacacacc acgccttcga gttcactgta acgacttcct 481 tattaagtcc atctgccgtg ccaactgctt gcgctacctc ttcttggctg agctgtttga 541 gctcaaagag gtatcagacg tagcttactc tggcattcgg gacaacttcc actactgggc 601 cagtcctgag ggctccatgc acttcatgcg ctgtccacct gttatctttg gccgcctgct 661 ccgtgatgaa aaccttcacg tgctcaatga agaccaggcg ctcagcgcac tcatcaattg 721 ggtgtacttc cggaaggagg atcgggagaa gtatttcaag aagttcttca attacatcaa 781 tctcagtgct gtctccaata agacgctggt gtttgccagc aacaagctgg tgggcatgga 841 gaacacctca tcccatacaa ccctgattga gagtgtcctg atggaccgca agcaggagcg 901 gccatgcagc ctgctggtct accagcggaa aggggccctg cttgattccg tggtcatcct 961 cggtggccag aaggcccacg gccagttcaa tgatggagtg tttgcttata tcatccagga1021 gaacctgtgg atgaagctct cagacatgcc ctatcgggca gcagcactta gtgccacctc1081 tgctggtcgc tacatctaca tctctggtgg caccactgag cagatttcag ggctgaagac1141 agcctggcgg tatgacatgg atgacaactc ctggaccaag ttgcctgacc tgcccatcgg1201 gcttgtcttc cacaccatgg tgacctgtgg ggggacagtg tactcagtgg gcgggagcat1261 tgccccaagg cggtatgtct ccaacatcta tcgctatgat gagcggaagg aagtctggtg1321 cctggcagga aagatgagca tccccatgga tggcaccgcc gtgatcacta aaggagacag1381 gcatctgtac attgtcactg gacggtgctt ggtgaaaggt tatatctccc gggtcggggt1441 agtggactgc tttgacacca gcactgggga cgtggtccag tgtatcacct tccccattga1501 gttcaaccat cggcccctgc tctctttcca acaggacaac atcctctgcg tgcacagcca1561 ccggcagagt gtggaaaaca atctgcagaa ggtgaaggca agcaagacga ccacctcagt1621 gcctgtcttg cccaacagct gccccttgga tgtgtcccat gctatatgct ccattggaga1681 cagcaaggtg tttgtatgtg ggggtgtcac cactgccagc gatgtccaga caaaggacta1741 caccatcaat ccaaatgcct tcttgctgga ccaaaagaca ggcaagtgga agaccctggc1801 tcctccacca gaggcactgg actgtcctgc ctgctgtcta gccaagctac cttgcaagat1861 tcttcaaagg atttaaacat tttaagtggg agaataagta aatgcattat tattcacgat1921 ttaatgagaa aaaaaaaaaa aaa
People's testicular sperm skelemin gene cDNA sequence
MKLEFTEKNYNSFVLQNLNRQRKRKEYWDMALSVDNHVFFAHRNVLAAVSPLVRSLISSNDMKTADELFITIDTSYLSPVTVDQLLDYFYSGKVVISEQNVEELLRGAQYFNTPRLRVHCNDFLIKSICRANCLRYLFLAELFELKEVSDVAYSGIRDNFHYWASPEGSMHFMRCPPVIFGRLLRDENLHVLNEDQALSALINWVYFRKEDREKYFKKFFNYINLSAVSNKTLVFASNKLVGMENTSSHTTLIESVLMDRKQERPCSLLVYQRKGALLDSVVILGGQKAHGQFNDGVFAYIIQENLWMKLSDMPYRAAALSATSAGRYIYISGGTTEQISGLKTAWRYDMDDNSWTKLPDLPIGLVFHTMVTCGGTVYSVGGsIAPRRYVSNIYRYDERKEVWCLAGKMSIPMDGTAVITKGDRHLYIVTGRCLVKGYISRVGVVDCFDTSTGDVVQCITFPIEFNHRPLLSFQQDNILCVHSHRQSVENNLQKVKASKTTTSVPVLPNSCPLDVSHAICSIGDSKVFVCGGVTTASDVQTKDYTINPNAFLLDQKTGKWKTLAPPPEALDCPACCLAKLPCKILQRI
People's testicular sperm skelemin gene open reading frame aminoacid sequence
The objective of the invention is: in view of human sperm skelemin 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 intratesticular spermatogeny 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 sperm skelemin gene encoded protein 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 people's testicular sperm skelemin abnormal gene 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.
Technical solution of the present invention
1. the preparation of human sperm skelemin gene fusion rotein
Nucleotide sequence and plasmid pDEST with people's testicular sperm skelemin gene open reading frame
TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, cultivate 0.5OD600 for 30 ℃ 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 sperm skelemin gene encoded 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 sperm skelemin gene encoded protein monoclonal antibody specific
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-people's testicular sperm skelemin antibody of secretion.
4. human sperm skelemin gene is used to prepare the testicular function gene expression chip
(1) with the nucleotide sequence design primer of the human sperm skelemin gene that obtains, the human sperm skelemin 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 sperm skelemin 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 sperm skelemin gene protein expression level of hybridization signal or the disappearance of human sperm skelemin gene.
Claims (5)
1. human sperm skelemin gene (CALICIN) is cloned the fusion of preparation; CALICIN,：MKLEFTEKNYNSFVLQNLNRQRKRKEYWDMALSVDNHVFFAHRNVLAAVSPLVRSLISSNDMKTADELFITIDTSYLSPVTVDQLLDYFYSGKVVISEQNVEELLRGAQYFNTPRLRVHCNDFLIKSICRANCLRYLFLAELFELKEVSDVAYSGIRDNFHYWASPEGSMHFMRCPPVIFGRLLRDENLHVLNEDQALSALINWVYFRKEDREKYFKKFFNYINLSAVSNKTLVFASNKLVGMENTSSHTTLIESVLMDRKQERPCSLLVYQRKGALLDSVVILGGQKAHGQFNDGVFAYIIQENLWMKLSDMPYRAAALSATSAGRYIYISGGTTEQISGLKTAWRYDMDDNSWTKLPDLPIGLVFHTMVTCGGTVYSVGGSIAPRRYVSNIYRYDERKEVWCLAGKMSIPMDGTAVITKGDRHLYIVTGRCLVKGYISRVGVVDCFDTSTGDVVQCITFPIEFNHRPLLSFQQDNILCVHSHRQSVENNLQKVKASKTTTSVPVLPNSCPLDVSHAICSIGDSKVFVCGGVTTASDVQTKDYTINPNAFLLDQKTGKWKTLAPPPEALDCPACCLAKLPCKILQRI
2. according to the described polyclonal antibody at the preparation of CALICIN 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 CALICIN 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 CALICIN gene cDNA according to claim 1 is described at CALICIN 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 CALICIN gene open reading frame aminoacid sequence, it is characterized in that fusion rotein can be used as the medicine of preparation treatment relative disease.
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| CN 01113507 CN1318559A (en) | 2001-04-11 | 2001-04-11 | Human sperm skelemin gene encoded protein |
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| CN 01113507 CN1318559A (en) | 2001-04-11 | 2001-04-11 | Human sperm skelemin gene encoded protein |
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2001
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