日本乙型脑炎病毒NS3蛋白的真核表达及鉴定

Eukaryotic expression and identification of Japanese encephalitis virus( JEV) NS3 protein

为了探讨日本乙型脑炎病毒(Japanese encephalitis virus,JEV)的复制机制及其蛋白与宿主蛋白的相互作用关系,试验以GenBank中收录的JEV HW1株NS3基因序列为模板设计引物,经PCR扩增获得NS3基因序列后,采用ClonExpress克隆技术将NS3基因连接到真核表达载体p EGFP-C3上,将得到的产物pEGFP-C3-NS3送生工生物工程(上海)股份有限公司测序;使用X-tremeGENE HP DNA Transfection Reagent体外转染幼地鼠肾细胞(BHK21细胞),观察24 h后应用Western-blot和IFA法进行鉴定。结果表明:PCR克隆获得的基因片段大小约为1 370 bp,NS3基因片段与HW1株同源性为100%;BHK21细胞密度在70%~80%时质粒转染效果最佳,质粒质量与转染试剂体积的比例为1∶3;Western-blot鉴定融合蛋白成功表达,分子质量约为79 ku;IFA鉴定融合蛋白成功表达,24 h为转染高峰期,绿色荧光最强,pEGFP-C3-NS3与NS3阳性血清反应显红色荧光。说明在BHK21细胞中成功表达出NS3蛋白。

In order to explore the replication mechanism of Japanese encephalitis virus(JEV)and the relationship between its protein and host protein,the primers were designed using the NS3 gene sequence of JEV HW1 strain included in GenBank as template.After amplifying the NS3 gene sequence by PCR,the NS3 gene was cloned into the eukaryotic expression vector p EGFP-C3 by ClonExpress cloning technology,and the obtained product pEGFP-C3-NS3 was sequenced by Sangon Biotech(Shanghai)Co.,Ltd..X-treme GENE HP DNA Transfection Reagent was used to transfect hamster kidney cell line(BHK21 cells)in vitro.After 24 hours of observation,the cells were identified by Western-blot and IFA.The results showed thathe size of the gene fragment cloned by PCR was about 1 370 bp,and the homology of NS3 gene fragment with HW1 strain was 100%.When the density of BHK21 cells was 70%-80%,the plasmid transfection was the best,and the ratio of plasmid mass to the volume of transfection reagent was 1∶3.Western-blot analysis showed that the fusion protein was successfully expressed,with molecular weight of about 79 ku.The fusion protein was successfully expressed by IFA.At this time,it was the peak of transfection,and the green fluorescence was the strongest.The reaction of pEGFP-C3-NS3 and NS3 positive serum showed red fluorescence.The results indicated that NS3 protein was successfully expressed in BHK21 cells.