猪日本乙型脑炎病毒NS5原核表达和多克隆抗体制备  

Prokaryotic expression and polyclone antibody preparation of nonstructural protein 5 of Japanese encephalitis virus from pig

作　　者：余风艳[1] 韩秀杰[1] 沈红霞[1] 张保新[1] 赵凡凡[1] 王晓杜[1] 

机构地区：[1]浙江农林大学动物科技学院,浙江临安311300

出　　处：《福建农林大学学报（自然科学版）》2013年第3期307-310,共4页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：浙江省自然科学基金资助项目(Y3110124);浙江农林大学人才启动基金项目(2010FR080)

摘　　要：提取猪日本乙型脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,扩增JEV-NS5基因片段,亚克隆到原核表达载体pET-28(a)上,构建重组原核表达质粒pET-28(a)-JEV-NS5,转化大肠埃希氏菌BL21(DE3)菌株,IPTG诱导表达重组JEV-NS5蛋白,获得分子质量为103 ku的重组蛋白.该蛋白主要以包涵体形式表达,表达量占总菌体蛋白的35%以上,His-band Ni+纯化后,获得高纯度重组蛋白占总蛋白的比例达75%以上.以纯化的蛋白免疫小鼠,制备小鼠抗JEV-NS5抗体,抗体效价达到3×104,并能与病毒感染的样本反应,证明该蛋白具有较好的特异性.The cDNA of Japanese encephalitis virus （JEV） was synthensized from viral genome by RT-PCR. The JEV-NS5 gene was cloned from cDNA by PCR. The JEV-NS5 gene was subcloned into pET-28（a） plasmid. The recombinant plasmid pETo28（ a）- JEV-NS5 was transformed into E. coll. BL21 （ DE3 ）, The recombinant JEV-NS5 protein was expressed by IFI＇G induction. SDS- PAGE results showed that its molecular weight is 103 ku. The protein was mainly expressed in the form of inclusion bodies. The re- combinant JEV-NS5 was more than 35% of the total cell protein by TLC scanning analysis. A large number of high-purity recombi- nant proteins were obtained by his-band Ni＋ purification. The proportion of recombinant JEV-NS5 reached more than 75% after pur- ification. The recombinant JEV-NS5 proteins were immunized into mouse, and mouse anti-JEV NS5 antibody was prepared. The an- tibody titer is 3 × 104. The specific of antibody was detected by westem-blottlng. This study was a tool and foundation d JEV NS.5 function and virus replication mechanism.

关 键 词：日本乙型脑炎病毒 非结构蛋白NS5 原核表达 纯化 

分 类 号：S855.3[农业科学—临床兽医学]