兔出血症病毒NSP1的原核表达及亚细胞定位

Prokaryotic expression and subcellular localization of rabbit hemorrhagic disease virus NSP1

为了研究兔出血症病毒非结构蛋白1(nonstructural protein 1,NSP1)的生物学特性,构建了含有NSP1基因的原核重组表达质粒pGEX-NSP1和与增强型绿色荧光蛋白融合表达的真核重组表达质粒pEGFP-NSP1;原核诱导表达NSP1蛋白,并对其进行纯化;同时利用脂质体介导方法将真核重组表达质粒瞬时转染兔肾细胞系,4,6-二脒基-2-苯基吲哚染细胞核后激光共聚焦观察NSP1在细胞内的表达和亚细胞定位情况。结果显示,原核诱导表达了含有GST标签的GST-NSP1蛋白,形成包涵体,大小约42ku;并且NSP1与增强型绿色荧光蛋白融合的蛋白定位于细胞质内。试验结果为深入探讨NSP1的生物学功能奠定了基础。

To study the biological characteristics of nonstructural protein 1（NSP1） of rabbit hemor- rhagic disease virus（RHDV）,RHDV NSP1 gene was amplified and cloned into pGEX-4T-1 and pEGFP-C3,respectively. The recombinant plasmids were named as pGEX-NSP1 and pEGFP-NSP1, respectively. The recombinant GST-NSP1 protein was expressed well in Escherichia coli by induction of IPTG （1 mmol/L） ,and the expressed NSP1 protein was purified. RK13 cells were transfected with the eukaryotic expression plasmid fused with the enhanced green fluorescent protein. Finally the expression of NSP1 in RK13 cells was observed by using laser confocal fluorescence microscope. The results indicated that the lo- calization of NSP1 protein was particularly in the cytoplasm,which laid the solid foundation for the further study on biological function of NSP1.