水貂阿留申病毒VP2基因的原核表达及初步应用  

Expression and Primary Application of Main Antigenic Region of VP2 Gene of Aleutian Mink Disease Parvovirus in Prokaryotic Expression System

作　　者：徐磊[1] 闫喜军[1] 张蕾[1] 柴秀丽[1] 赵建军[1] 张海玲[1] 高晗[1] 白雪[1] 

机构地区：[1]中国农业科学院特产研究所,长春130122

出　　处：《中国农学通报》2010年第8期7-11,共5页Chinese Agricultural Science Bulletin

基　　金：科技部科研院所社会公益研究专项"野生动物重要传染病生态学发生;监测和控制技术"(2005DIB4J048)

摘　　要：将水貂阿留申病毒ADV-G株VP2基因中主要抗原表位区的2个片段,分别克隆至原核表达载体pET-28a(+)的多克隆位点中,鉴定后得到重组质粒pET-VP2a和pET-VP2b,将重组质粒转化到宿主菌BL21中,用IPTG分别以不同浓度,不同诱导时间进行诱导表达,采集样品做SDS-PAGE、Western-blot分析,并以此蛋白作为包被抗原进行ELISA检测。结果发现以终浓度为1mmol/L的IPTG进行诱导,4h后表达可达到高峰,其大小分别约为23kD和28kD,该蛋白与ADV阳性血清能发生特异性反应。将此表达产物应用Ni-NTAHis-Bind纯化系统进行纯化,其纯度可达到91%,纯化后的蛋白以60μg/孔包被96孔板,检测不同地区水貂血清,同时以对流免疫电泳法(CIEP)为对照,结果发现二者的符合率高达93%,而应用该蛋白进行检测其反应更为安全,背景低,制备简单,这为今后应用该蛋白作诊断抗原检测水貂阿留申病奠定了基础。The recombinant expression vectors pET-VP2a and pET-VP2b were constructed by cloning VP2 gene of Aleutian mink disease parvovirus （ADV-G） into a prokaryotic expression plasmid pET-28a. The recombinant vectors were transformed into recipient germs BL21. Samples were collected at different induction time after induction with IPTG. The specificity of the expressed proteins were identified with SDS-PAGE, Western Blotting . And the protein were about 23kDa and 28 KDa in size. The expressed protein were purified byNi-NTA His-Bind purification system and the degree of purity is about 91%.The purified protein was coated on 96-well plate at 60μg/well and used for identification of ADV-positive sera from different regions. The results are 93% identical to CIEP. But this method is safer and easier to perform. High efficient expression of ADV protein will benefit for diagnosis of ADV in practice.

关 键 词：水貂阿留申病毒 VP2基因 原核表达 

分 类 号：S858.953[农业科学—临床兽医学]