乙型脑炎病毒SA14-14-2株E蛋白C端片段的克隆及原核表达

目的]为进一步研制检测乙脑抗体试剂盒奠定了基础。方法]以本实验室已构建的重组质粒pET-E(E为乙脑疫苗株SA14-14-2株E蛋白)为模板,利用PCR扩增乙型脑炎病毒E蛋白基因3′端部分片段,克隆入原核表达载体pET-32a(+),转化大肠杆菌BL21(DE3),经IPTG诱导表达后,SDS-PAGE电泳分析表达产物。结果]所表达的融合蛋白分子量约为38KD,表达产物以水溶形式存在,37℃,诱导4 h的诱导培养条件最佳,表达量约占茵体总蛋白的20?。表达产物经Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白。ELISA检测表明,表达的蛋白能与猪乙脑病毒抗体血清发生特异性反应,具有很好的抗原性。结论]基因工程表达的JEV E蛋白有望成为重要的实验室诊断抗原。

Clone of C-terminal Fragment of SA14-14-2 Strain of Japanese Encephalitis Virus E Protein and its Expression in Escherichia coli

Objective] The aim was to lay a foundation for developing JEV antibody test kit.Method] 3-terminal Fragment of SA14-14-2 Strain of Japanese encephalitis virus E protein was amplified from the recombinant plasmid pET-E(E was SA14-14-2 strain of Japanese encephalitis virus E protein) constructed by this laboratory.The fragment was inserted into pET-32a(+) expressive vector,then transformed into Escherichia coli BL-21(DE3),and then the expressive production was analyzed through the SDS-PAGE electrophoresis after induced by IPTG.Result] The molecular weight of the expressive fusion protein was 38 KD and the expressive production existed in the form of water solution.The optimum inducing cultivation condition was the temperature of 37 ℃ and the induction duration of 4 h.The expression shared 20% of the total protein of the strain.The target protein with high purity was obtained after the expressive production purified by Ni NTA affinity chromatography.ELISA detection showed that the expressive protein could react specifically with antibody serum of pig Encephalitis Virus and it had good antigenicity.Conclusion] The JEV E protein expressed by genetic engineering was hopeful to be an important laboratory diagnostic antigen.