To improve the effect of the gene immunization against Hantaan virus, we constructed the eukaryotic expression vector pTARGET-hans(ISS) containing Hantaan Virus S gene coding region and CpG motif by cloning S gene segment with CpG motif into eukaryotic expression vector pTARGETTM. After conformed by enzyme analysis, the recombinant expression vector pTARGET-hans(ISS) was transferred into Vero-E6 cells by electroporation and the transient expression of Hantaan virus nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transferred Vero-E6, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector pTARGET-hans(ISS) was successfully constructed and expressed in vitro, which will lay a foundation for further animal vaccination.
实验以汉坦病毒76-118株M基因为模板进行PCR扩增得到含完整编码汉坦病毒G2蛋白基因M2的1 600 bp DNA片段,将此基因片段重组到T载体pMD18中,转化至大肠埃希菌DH5α后,筛选阳性菌落扩增培养,提取重组质粒,双酶切鉴定后,克隆至表达载体pGEX-6P-1-M2,亲和层析法纯化融合蛋白,产物进行Western-Blot鉴定。实验证实通过以质粒pGEX-6P-1为载体,可构建编码汉坦病毒76-118株包膜糖蛋白G2的M2基因的原核表达载体pGEX-6P-1-M2,并可观察G2蛋白在原核细胞中的表达。
