流行性乙型脑炎病毒E基因的克隆、表达及初步应用.PDFVIP

流行性乙型脑炎病毒 E 基因的克隆、表达及初步应用 张雪寒 ,何孔旺 ,郭容利 ,倪艳秀 ,王 　芳 ,俞正玉 (江苏省农业科学院兽医研究所 农业部畜禽疫病诊断重点开放实验室 ,江苏南京 2 100 14) Cloning and Expression of E Gene of Japanese Encephal itis Virus and Prel iminary appl ication ZHAN G Xuehan , H E Kongwang , GU O Ron gli , N I Yanxiu ,WAN G Fan g , YU Zhengyu ( Key L ab of A ni m al Poul t ry D isease D i ag nos is , M i nis t ry of A g ricul t u re , I ns t it ute of Veteri nary S cience of J i angs u A ca de ) my of A g ricul t u ral S ciences N anj i ng 2 100 14 , Chi na Abstract : The w hole cDN A of E gene wa s amp lified by R TPCR f ro m J EV st rain SA 1414 , an d cloned into t he p MD 18T vector . The f ragment wa s identified by re st riction enzyme s dige stio n wit h EcoR I and X ho I ,cloned into t he p E T32a ( + ) vector . The reco mbinant p la smid wa s t ran s for med into BL 2 1 an d t he reco mbinant bact eria wa s induced by op timal concent ration of IP T G. SD SPA GE and We st er n blot ting were p erfor med to det ect t he E f u sion p rot ein . Our re sult in di cat e s t he molecular weight of E p rot ein i s of E p rot ein 73kDa and it s antigenicit y i s goo d . In direct EL ISA p rocedure ha s been develop ed wit h t he p urified E p rot ein a s antigen for det ection of J EV . Key words : J ap anese encep h al i t is v i r us (J EV) ; E gene ; In direct EL ISA ( ) 摘要 : 参照 GenBank 中的日本乙型脑炎病毒 J ap anese encep hal i tis v i rus ,J EV SA 1414 株序列设计了一对特异性 引物 ,用 PCR 方法从 SA 1414 扩增 E 基因全长 ,然后克隆到p MD 18T 载体中 ,转化宿主菌D H5a ,提取阳性克隆质 粒进行双酶切鉴定 ,将 目的片段定向克隆到 p E T32a ( + ) 中 ,转化入 BL 2 1 (D E3) ,经 IP T G 诱导可