鸭圆环病毒orf3基因的序列研究及基因表达-sequence study and gene expression of duck circovirus or f3 gene.docxVIP

鸭圆环病毒 ORF3 基因的序列分析及基因表达baculovirus expression systems to construct the recombinant transfer vector pFast-ORF3. The transfer vector was transformed into DH10Bac competent cell which contain the BmNPV bacmid. A recombinant shuttle vector which contain ORF3 gene was constructed by site- specific transposition and the colonies containing recombinant bacmid were collected by white selection. The cultured sf9 cells were transfected with recombinant bacmid DNA mediating with cellfectin reagent and the pure recombinant baculovirus Bacmid-ORF3 was obtained. After transfecting this transformant into Sf9 cells, ORF3 protein expression in Sf9 cells was detected by western blotting. The experimental results showed that the recombinant buculovirus was obtained and the cap protein with a molecular weight of 17kD was expressed in insect Sf9 cells. The recombinant protein can be recognized by DuCV-positive serum. This would lay a foundation for developing DuCV.Key words: duck circorirus; ORF-3; sequence analysis; genotype;prokaryotic expression; Eukaryotic expression符号及缩略词英文缩写英文全称中文全称DNADeoxyribonucleic acid脱氧核糖核酸PCRPolymerase chain reaction聚合酶链反应dNTPDeoxyribonuleoside triphosphate脱氧核糖核苷三磷酸SDSSodium dodecyl sulfate十二烷基硫酸钠PAGEPolyacrylamide gel electrophoresis聚丙烯酰胺凝胶电泳AmpAmpicillin氨苄青霉素LBODLuria-bertani mediumOptical dendityLB-培养基光密度bpBase pair碱基对kDkilodalton千道尔顿rpm IPTGTMBRevolutions per minute Isopropyl-β-D-thiogalactosideTetramethyl benzidine转速/分钟异丙基-β-D-半乳糖苷 四甲基联苯胺FBSFetal bovine serum胎牛血清TEMEDLNNNN’-tetramethyl aminomethaneLiterNNNN’-四甲基氨基甲烷升AAAmino asides氨基酸关于学位论文原创性和使用授权的声明本人所呈交的学位论文，是在导师指导下，独立进行科学研究所取得 的成果。对在论文研究期间给予指导、帮助和做出重要贡献的个人或集 体，均在文中明确说明。本声明的法律责任由本人承担。本人完全了解山东农业大学有关保留和使用学位论文的规定，同意学 校保留和按要求向国家有关部门或机构送交论文纸质本和电子版，允许论 文被查阅和借阅。本人授权山东农业大学可以将本学位论文的全部或部分 内容编入有关数据库进行检索，可以采用影印、缩印或其他复制手段保存 论文和汇编本学位论文，同时授权中国科学技术信息研究所将本学位论文 收录到《中国学位论文全文数据库》，并向社会公众提供信息服务。保密论文在解密后应遵守此规定。论文作者签名： 导 师 签 名： 日期：1 引 言圆环病毒属于圆环病毒科（Circoviridae），是一类无囊膜单链环状 DNA 病毒，分 为两个属，分别是环病毒属（Gyrovirus）和圆环病毒属（Circovirus）（To