CN1319665A - Human testics kinesin 9 gene coding protein - Google Patents

Abstract

The present invention belongs to the field of human genome technology. It relates to human testis kinesin protein 9 gene, its total length cDNA sequence is 2510 bp, its open reading frame is 2178 bp, it codes 726 amino acids, the serial number of said gene in gene bank is AF311212. The invented human testis kinesin protein gene can be used to express clone, can be used for preparing fusion protein, said protein can be used to immunize animal to prepare monoclonal antibody and polyconal antibody, and the gene clone can be used for preparing testis specific function gene expression chip.

Description

KINESIN PROTEIN 9's gene coded protein

The invention belongs to the Human genome technical group field.

KINESIN PROTEIN 9's gene of the present invention 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 full-length cDNA through sequencing, be 2510bp; Its open reading frame is 2178bp, its 726 amino acid of encoding.Consult gene pool, this gene and mouse kinesin 9 dna homologs, the mankind do not see homologous gene.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 KINESIN PROTEIN 9's gene of finding is relevant with spermatogeny probably, use this gene clone, can be prepared into fusion rotein, 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.

Because KINESIN PROTEIN 9's 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 Center for BiotechnologyInformation NCBI ) formally accepts the newcomer that this gene is Genbank, and accepting number is AF311212 in American National biotechnology information center.1 ggaggtggtt tcggttgcgg cagtcgcgtc ccgggagcgt cgctgcctgg tgaacgctag 61 aatgggtact aggaaaaaag ttcatgcatt tgtccgtgtc aaacccaccg atgactttgc 121 tcatgaaatg atcagatacg gagatgacaa aagaagcatt gatattcact taaaaaaaga 181 cattcggaga ggagttgtca ataaccaaca gacagactgg tcgtttaagt tggatggagt 241 tcttcacgat gcctcccagg acttggttta tgagacagtt gcaaaggatg tggtttctca 301 ggccctcgat ggctataatg gcaccatcat gtgttatggg cagacgggag ctggcaagac 361 atacaccatg atgggggcaa ctgagaatta caagcaccgg gggatcctcc ctcgtgccct 421 gcagcaggtt tttaggatga tcgaagaacg ccccacacat gccatcactg tgcgtgtttc 481 ctacttggaa atctataatg agagcctgtt tgatctcctg tccactctgc cctatgttgg 541 accctcagtc acaccaatga ccatcgtgga aaaccctcaa ggagtcttca ttaagggctt 601 gtcagttcac ctcacaagtc aggaggagga tgcattcagc ctcctttttg agggtgagac 661 caacaggatt atagcctccc acactatgaa caaaaactct tccagatcac actgcatttt 721 caccatctac ttagaggccc attcccggac cttatcagag gaaaagtaca tcacttccaa 781 aattaacttg gtggatctgg caggctcaga gaggctgggg aagtctgggt ctgagggcca 841 agtcctgaag gaagccacct acatcaacaa atcgctctca ttcctggagc aggccatcat 901 tgcccttggg gaccagaagc gggaccacat cccctttcgg cagtgcaagc tcacccacgc 961 tctgaaggac tcgttagggg gaaactgcaa tatggtcctc gtgacaaaca tctatggaga1021 agctgcccag ttagaagaaa cgctatcttc actgagattt gccagcagga tgaagctagt1081 caccactgag cctgccatca atgaaaagta tgatgctgag agaatggtca agaacctgga1141 gaaggaacta gcactactca agcaggagct ggctatccat gacagcctga ccaaccgcac1201 ctttgtgacc tatgacccca tggatgaaat ccagattgct gagatcaact cccaggtgcg1261 gaggtacctg gaggggacac tggacgagat cgacataatc agccttagac agatcaagga1321 ggtgttcaac cagttccggg tggttctgag ccaacaggaa caggaagtgg agtccacttt1381 gcgcaggaag tacaccctca ttgacaggaa tgactttgca gccatttctg ctatccagaa1441 ggcggggctt gtggatgttg atggccacct agtgggtgag cctgaaggac aaaactttgg1501 actcggagtc gcccctttct ctaccaaacc tgggaagaaa gccaagtcca agaagacatt1561 caaagagcca ctcaggcccg acaccccacc ctccaaacca gtggcctttg aggagtttaa1621 gaatgagcaa ggtagtgaga tcaaccgaat tttcaaagaa aacaaatcca tcttgaatga1681 acggaggaaa agggccagcg agaccacaca gcacatcaat gccatcaagc gggagattga1741 tgtgaccaag gaggccctga atttccagaa gtcactacgg gagaagcaag gcaagtacga1801 aaacaagggg ctgatgatca tcgatgagga agaattcctg ctgatcctca agctcaaaga1861 cctcaagaag cagtaccgca gcgagtacca ggacctgcgt gacctcaggg ctgagatcca1921 gtattgccag cacctagtgg atcagtgtcg ccaccgcctg ctcatggaat ttgacatctg1981 gtacaatgag tcctttgtca tccctgagga catgcagatg gcactgaagc caggcggcag2041 catccggcca ggcatggtcc ctgtgaacag gattgtgtct ctgggagaag atgaccagga2101 caaattcagc cagctgcagc agagggtgct tcctgagggc cctgattcca tctccttcta2161 caatgccaaa gtcaagatag agcagaagca taattacttg aaaaccatga tgggcctcca2221 gcaggcacat agaaaataga acctcatcgc cagtacctta aaggacaaga ccagcaactc2281 ccacctactg tagtggagct gctcaaccac ctgcccagag ctgcagcccc ctgtactcca2341 atgctgggac ccagcacagc gaacacattt ggcctgcatg ttgggaggag catcctccaa2401 ggacaacctt gctcatctcc acagagcact ttgggtttta attcactgtc ttatatgcag2461 ggacaggata aaataacttt ctagtttgga cttttaaaaa aaaaaaaaaa9cDNAMGTRKKVHAFVRVKPTDDFAHEMIRYGDDKRSIDIHLKKDIRRGVVNNQQTDWSFKLDGVLHDASQDLVYETVAKDVVSQALDGYNGTIMCYGQTGAGKTYTMMGATENYKHRGILPRALQQVFRMIEERPTHAITVRVSYLEIYNESLFDLLSTLPYVGPSVTPMTIVENPQGVFIKGLSVHLTSQEEDAFSLLFEGETNRIIASHTMNKNSSRSHCIFTIYLEAHSRTLSEEKYITSKINLVDLAGSERLGKSGSEGQVLKEATYINKSLSFLEQAIIALGDQKRDHIPFRQCKLTHALKDSLGGNCNMVLVTNIYGEAAQLEETLSSLRFASRMKLVTTEPAINEKYDAERMVKNLEKELALLKQELAIHDSLTNRTFVTYDPMDEIQIAEINSQVRRYLEGTLDEIDIISLRQIKEVFNQFRVVLSQQEQEVESTLRRKYTLIDRNDFAAISAIQKAGLVDVDGHLVGEPEGQNFGLGVAPFSTKPGKKAKSKKTFKEPLRPDTPPSKPVAFEEFKNEQGSEINRIFKENKSILNERRKRASETTQHINAIKREIDVTKEALNFQKSLREKQGKYENKGLMIIDEEEFLLILKLKDLKKQYRSEYQDLRDLRAEIQYCQHLVDQCRHRLLMEFDIWYNESFVIPEDMQMALKPGGSIRPGMVPVNRIVSLGEDDQDKFSQLQQRVLPEGPDSISFYNAKVKIEQKHNYLKTMMGLQQAHRK9

The objective of the invention is: in view of KINESIN PROTEIN 9's 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, therefore this gene is relevant with intratesticular spermatogeny probably, its unconventionality expression may influence the male sex's reproductive function, therefore the research of this gene can be used for: 1. by this gene Fusion albumen of preparation, be used to study the relation of 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 KINESIN PROTEIN 9's gene coded 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 the white abnormal gene expression relative disease of human testics kinesin 9 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 proteic preparation of the white gene fusion of technical solution of the present invention 1. human testics kinesin 9s

Nucleotide sequence and plasmid pDEST with KINESIN PROTEIN 9's gene open reading frame ^TM15 are prepared into the GST-SP2-pDEST15 expression plasmid, cultivate 0.5 OD600 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. KINESIN PROTEIN 9's 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. KINESIN PROTEIN 9's gene coded 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-KINESIN PROTEIN 9's gene of secretion.4. KINESIN PROTEIN 9's gene is used to prepare the testicular function gene expression chip

(1) with the nucleotide sequence design primer of KINESIN PROTEIN 9's gene of obtaining, the white gene order of human testics kinesin 9 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 33P mark of KINESIN PROTEIN 9's genetically deficient:

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 white expression of gene protein level of strong and weak analyzing influence human testics kinesin 9 of hybridization signal or the disappearance of KINESIN PROTEIN 9's gene.

Claims (4)

1. a sperm function specific gene is cloned the fusion for preparing; 9,：MGTRKKVHAFVRVKPTDDFAHEMIRYGDDKRSIDIHLKKDIRRGVVNNQQTDWSFKLDGVLHDASQDLVYETVAKDVVSQALDGYNGTIMCYGQTGAGKTYTMMGATENYKHRGILPRALQQVFRMIEERPTHAITVRVSYLEIYNESLFDLLSTLPYVGPSVTPMTIVENPQGVFIKGLSVHLTSQEEDAFSLLFEGETNRIIASHTMNKNSSRSHCIFTIYLEAHSRTLSEHKYITSKINLVDLAGSERLGKSGSEGQVLKEATYINKSLSFLEQAIIALGDQKRDHIPFRQCKLTHALKDSLGGNCNMVLVTNIYGEAAQLEETLSSLRFASRMKLVTTEPAINEKYDAERMVKNLEKELALLKQELAIHDSLTNRTFVTYDPMDEIQIAEINSQVRRYLEGTLDEIDIISLRQIKEVFNQFRVVLSQQEQEVESTLRRKYTLIDRNDFAAISAIQKAGLVDVDGHLVGEPEGQNFGLGVAPFSTKPGKKAKSKKTFKEPLRPDTPPSKPVAFEEFKNEQGSEINRIFKENKSILNERRKRASETTQHINAIKREIDVTKEALNFQKSLREKQGKYENKGLMIIDEEEFLLILKLKDLKKQYRSEYQDLRDLRAEIQYCQHLVDQCRHRLLMEFDIWYNESFVIPEDMQMALKPGGSIRPGMVPVNRIVSLGEDDQDKFSQLQQRVLPEGPDSISFYNAKVKIEQKHNYLKTMMGLQQAHRK

2. according to the described polyclonal antibody at the preparation of KINESIN PROTEIN 9's 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 KINESIN PROTEIN 9's 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 KINESIN PROTEIN 9 gene cDNA according to claim 1 is described at KINESIN PROTEIN 9's 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.