返回 2008年第31卷第3期 目录

流行性乙型脑炎病毒E蛋白基因在大肠杆菌中的克隆与表达*

王 丽，李 洪，张志霞，刘晓燕，谢华福，宋 杰，梅志强

(泸州医学院医学分子生物学实验室，四川泸州 646000)

摘 要 目的； 获得JEV E蛋白基因，构建重组表达载体并在E.coli中高效表达。方法：参照GeneBank中的日本乙型脑炎病毒SA14-14株序列设计一对特异性引物，采用RT-PCR法扩增E基因全长，克隆至PUCm-T载体，经测序证实后克隆至原核表达载体pET32a中，转化入BL21（DE3），IPTG诱导表达，对表达产物进行SDS-PAGE电泳分析。结果: 限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明，扩增出了JEV E蛋白基因全长1500bp，成功构建了重组质粒pET-32a/JEV E，SDS-PAGE分析显示目的蛋白主要以包涵体的形式在大肠杆菌中获得表达，表达量占总菌体蛋白的35 %。结论: 在大肠杆菌中成功表达了JEV E蛋白，为ELISA早期诊断试剂盒的研制和E蛋白基因结构与功能的分析奠定了基础。

关键词 流行性乙型脑炎病毒；E蛋白基因；反转录-聚合酶链反应；基因克隆；表达载体

*四川省教育厅科研基金课题（项目编号：2006ZD019）

CLONING AND EXPRESSION OF JAPANESE ENCEPHALITIS VIRUS E

GENE IN ESCHERICHIA COLI

Wang Li, et al

Laboratory of Medical Molecular Biology, Luzhou Medical College

Abstract Objective: To construct a fused expression vector of JEV gene and make it has a high expressied in E.coli. Methods: The whole cDNA of JEV E gene was amplified by RT-PCR from JEV strain SA14-14 and cloned into PUCm-T vector. After it was verified through DNA sequencing analysis, the fragment was cloned into pET32a expression vector. Then the recombinant plasmid was transformed into BL21 cells and induced with isoproply-β-D-thiogalactoside (IPTG)to express. The expression product was identified with SDS-PAGE. Results:By restriction endonuclease digestion, PCR and DNA sequencing analysis, the cDNA fragments of JEV E gene consisting of 1500 nucleotides were successfully cloned into PUCm-T and pET32a vectors respectively. The expression E protein band with a relative molecular mass of 52kDa was detected by SDS-PAGE. The target protein mainly in the form of inclusion body was highly expressed in E.coli and amounted to 35 % by total protein. Conclusion: The recombinant JEV E protein has been expressed in E.coli successfully,which lays a good foundation for future researches on its structure and function as well as the developing of Kit ELISA for the early diagnosis of epidemic encephalitis.

Key words Japanese encephalitis virus; JEV E gene; RT-PCR; Gene cloning; Expression vector