日本脑炎病毒E基因抗原域Ⅲ的克隆及高效表达  

Cloning and expression in E.coli for the domain Ⅲ of the envelope protein gene of the attenuated SA14-14-2 strain of Japanese encephalitis virus

作　　者：郑其升[1] 杨松 徐学清[1] 曹瑞兵[1] 陈德胜[1] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,江苏南京210095

出　　处：《南京农业大学学报》2004年第4期77-80,共4页Journal of Nanjing Agricultural University

基　　金：国家高技术发展计划资助项目 (2 0 0 1AA2 490 12 )

摘　　要：根据GenBank公布的日本脑炎病毒 (Japaneseencephalitisvirus ,JEV)SA14 14 2减毒株的核苷酸序列 ,设计并合成一对特异性引物 ,应用RT PCR方法扩增出编码JEV囊膜蛋白抗原域Ⅲ的基因 (EⅢ) ,将其连接入pMD18 T载体 ,经测序后克隆入原核表达载体pET 32a (+) ,构建原核表达载体pET EⅢ 。阳性质粒转化感受态大肠杆菌BL2 1(DE3) ,经IPTG诱导 ,JEV EⅢ 基因获得表达 ,经细胞定位分析 ,目的蛋白以包涵体的形式存在于大肠杆菌中。通过改变IPTG的浓度和诱导时间 ,确定了表达EⅢ 蛋白的最佳诱导条件 :IPTG终浓度为 0 1mmol·L-1,诱导时间为 3h。在大肠杆菌中的最高表达量占菌体总蛋白的 5 4 9%。Western blot分析表明表达产物具有良好的免疫学活性。利用纯化的表达产物作抗原包被酶标板 ,可以明显地区分JEV阴、阳性血清 ,并且不与猪伪狂犬病病毒、猪圆环病毒、猪呼吸与繁殖障碍综合征病毒、猪细小病毒、猪瘟病毒的阳性血清发生反应。结论 :重组JEVEⅢ 蛋白可以用于检测猪血清中JEV抗体水平。Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank,the domain Ⅲ of the E gene from Japanese encephalitis virus(JEV)attenuated strain SA14-14-2 was amplified by RT-PCR method.The RT-PCR product was successively cloned into pMD18-T vector and pET-32a(+)to get a recombinant prokaryotic expression plasmid pET- E Ⅲ.The target gene was expressed in the inclusion body in BL21(DE3)when induced with IPTG.The expression was optimized with the proper inducing conditions of 0.1 mmol·L -1 IPTG and 3 hours induction.The highest expression of the target protein accounts to 54.9% of E.coli total proteins.Western-blot analysis showed that the expression product had immunogencity.In indirect ELISA,the target protein can distinctly differentiate JEV positive serum from JEV negative serum.Furthermore,The recombinant E Ⅲ protein does not react with the positive serum of other reproductive diseases such as PRRSV,CSFV,PRV,PPV and HCV. Conclusions:The E Ⅲ can be applied in the detection for JEV antibodies in swine serum.

关 键 词：日本脑炎病毒 囊膜蛋白 克隆 原核表达 

分 类 号：S852.43[农业科学—基础兽医学]