西尼罗病毒糖蛋白第三结构域的原核表达及鉴定

Prokaryotic expression and identification of West Nile virus EDⅢ gene

根据西尼罗病毒(West Nile virus,WNV)序列设计1对特异性引物,用PCR反应扩增糖蛋白第三结构域(WNVEDⅢ)基因,将PCR产物克隆至pGEX-4T-1原核表达载体上,获得重组表达质粒,将该质粒转化入E.coli DH5α,命名为pG-EDⅢ,鉴定正确后转入transetta表达菌中,经IPTG诱导后纯化重组蛋白,通过SDS-PAGE和Western-blot方法证实WNV-EDⅢ基因可在大肠杆菌中高效表达,小鼠免疫试验证明该原核表达蛋白有良好的免疫原性。

A pair of primers was designed according to the reported WNV sequences and applied for amplifying the E protein domain Ⅲ （WNV-EDⅢ） fragment by PCR. The recombinant plas- mid was constructed by cloning the PCR product into thepGEX-4T-1 vector. Then the constructed recombinant plasmid was transformed into E. coli DH5a and named pG-EDⅢ. After identified, pG-EDⅢ was transformed into transetta competent cell,the recombinant protein was purified fol- lowed by IPTG induced. According to SDS-PAGE and western blot WNV-ED Ⅲ protein was ex- pressed at high level. The good immunogenicity of the recombination protein was proved through immunity test in mice.