乙型脑炎病毒NS5蛋白原核表达与亚细胞定位研究

Prokaryotic expression and subcellular localization of Japanese encephalitis virus NS5 protein

目的对乙型脑炎病毒NS5蛋白进行原核表达和亚细胞定位研究。方法通过PCR扩增乙型脑炎病毒NS5基因,并将之克隆至原核表达载体pET-28a(+)中,构建的重组表达载体转化大肠杆菌感受态细胞,经IPTG诱导表达目的蛋白。将表达产物纯化后,免疫BALB/c小鼠,制备NS5蛋白特异性的多克隆抗体。同时,构建NS5基因与绿色荧光蛋白(EG-FP)融合表达的真核表达质粒pcDNA-NS5-EGFP,将之转染BHK-21细胞,利用激光共聚焦荧光显微镜观察NS5蛋白在BHK-21细胞中的亚细胞定位。结果 NS5蛋白在大肠杆菌中获得了高效表达,表达蛋白分子量大小约为103kD,该蛋白在真核细胞中的表达呈颗粒状不均匀分布于整个细胞,且颗粒状主要分布在细胞质中。结论成功表达乙型脑炎病毒NS5蛋白,并对NS5蛋白的亚细胞定位进行了分析,为进一步研究NS5蛋白提供依据。

The NS5 gene of Japanese encephalitis virus was amplified by PCR and cloned into the prokaryotic expression vector pET-28a.The recombinant NS5 protein was expressed highly under induction of IPTG in the E.coli.Using BALB/c female mice immuned with the purified recombinant NS5 protein,NS5 protein-specific polyclonal antibody were prepared.Meanwhile,BHK-21 cells were transfected with eukaryotic expression plasmid pcDNA-NS5-EGFP of NS5 gene and green fluorescent protein（EGFP） fusion expression.Finally,the subcellular localization of NS5 protein in BHK-21 cells was observed by using laser confocal fluorescence microscope.The result indicates that the protein particles are irregularly distributed in the whole cell and particularly in the cytoplasm.