摘要
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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2011年第9期778-782,共5页
Chinese Journal of Zoonoses
关键词
NS5
原核表达
亚细胞定位
乙型脑炎病毒
NS5
prokaryotic expression
subcellular localization
Japanese encephalitis virus