摘要
根据GenBank公布的日本脑炎(Japanese encephalitis virus,JEV)SA14-14-2减毒株E基因的核苷酸序列,设计并合成一对特异性引物,采用RT-PCR方法扩增其E基因全长cDNA,将扩增产物克隆入pUCm-T载体中,测序后亚克隆到原核表达载体pET-32a(+),筛选重组质粒,转化大肠埃希菌BL21(DE3)宿主菌。经IPTG诱导,SDS-PAGE分析表达产物。结果获得了含全长日本脑炎病毒E基因的重组质粒,经测序证实,与GenBank上E基因序列的同源性达到100%。所表达的融合蛋白主要以包涵体形式存在,为制备JEV实验室诊断抗原打下了基础。
According to the eDNA sequence of JEV E protein derived from the GenBank （Accession No. AF495589） ,a pair of primers were designed. Genomie RNA was isolated from JEV attenuated strain SA14- 14-2,and used as templates for eDNA synthesis of E gene. Subsequently the eDNA encoded JEV E protein was synthesized by using RT-PCR. Then the amplified fragment was cloned into vector pUCm-T and transformed into E. coli TG1. After sequencing, the recombinant expression plasmid pET-32a（A-）/E was constructed and transformed into E. coli BL21（DE3）. After induced by IPTG,SDS-PAGE was performed to detect the E gene product. The E gene had the identies of 100% with the sequence of JEV E protein published in the GenBank（Aeeegsion No. AF495589）and the JEV E protein was expressed successfully in inclusion body,which should be useful for the production of diagnostic reagents.
出处
《动物医学进展》
CSCD
2007年第3期23-26,共4页
Progress In Veterinary Medicine
关键词
日本脑炎病毒
减毒活疫苗
E基因
表达
Japanese encephalitis virus
attenuated vaccine
E gene
expression
