乙型脑炎病毒E蛋白基因的克隆及其在大肠埃希菌中的表达

Gene cloning and expression of Japanese encephalitis virus E protein in Escherichia coli

目的 在大肠埃希菌中表达乙型脑炎病毒E蛋白。方法 反转录聚合酶链反应 (PCR)扩增乙型脑炎病毒 (JEV)E蛋白基因 ,克隆入原核表达载体pET 2 8a,转化大肠埃希菌BL2 1(ED3)。经异丙基巯基半乳糖 (IPTG)诱导表达后 ,十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白免疫印迹分析表达产物。结果 本室所保存毒株的E蛋白与已发表的序列比较 ,有 5个核苷酸改变 ,可分别导致氨基酸替代。所表达的E蛋白的相对分子质量约为 5 0 0 0 0 ,表达量约占菌体总蛋白的 35 %。结论 在大肠埃希菌中成功地表达了JEVE蛋白 。

Objective To express Japanese encephalitis virus (JEV) E protein in Escherichia coli. Methods The gene coding for JEV E protein was amplified by using RT-PCR, and cloned into plasmid pET-28a. The constructed plasmid was transformed into E.coli BL21 (DE3). Expression of E protein by the transformants was induced by IPTG and analyzed by SDS-PAGE and Western blot. Results Five nucleotide changes leading to amino substitutions were identify in the E protein gene of our laboratory strain of JEV when compared to a published gene sequence of JEV E protein. The yield of expressed JEV E protein with relative molecular mass approximately 52×10 3 was 35% of total bacterial proteins. Conclusions JEV E protein was expressed successfully in E.coli, which should be useful for the production of diagnostic reagents and the analysis of gene structure/function of the E protein.