鼻病毒非结构蛋白2B引起细胞自噬产生的研究

A autophagy induced by non-stuctural protein 2B of human rhinovirus

目的利用miniSOG标签观察鼻病毒非结构蛋白2B蛋白在细胞中定位及自噬体产生。方法将非结构蛋白2B和miniSOG进行融合表达构建pcDNA3.1-2B-miniSOG-flag质粒,将质粒转染HEK293细胞,在荧光显微镜下,2B-miniSOG阳性细胞进行固定、光氧化及超薄切片制备,电镜下观察2B-miniSOG蛋白在细胞中的定位及细胞超微结构变化,Westernblot(WB)检测LC3的表达。结果2B-miniSOG蛋白在荧光显微镜下发出绿色荧光,WB检出自噬标志性蛋白LC3-II表达增加。电镜下观察到蛋白定位于线粒体,细胞中出现大量囊泡样结构,可观察到自噬体和自噬溶酶体。结论鼻病毒非结构蛋白2B可引起细胞自噬发生。

Objective To study the intracellular location and autophagosome production of rhinovirus 16 2B protein using miniSOG labeling technique.Methods 2B was fused with miniSOG and flag tags to construct pcDNA3.1-2B-miniS0G-flag plasmid,which was used to transfect HEK293 cells,LC3 protein was detected by western blot.The transfected cells were fixed,stained with DAB through the photooxidation activity of miniSOG,and used to prepare ultrathin sections.Localization of 2B-miniS0G protein in cells and ultrastructural changes of cells were observed under electron microscope.Results 2B-miniSOG protein glows green under a fluorescence microscopy.Green flourescence coold be observed in the cells expressing 2B-miniSOG protein.LC-II protein increased in the cells transfected with pcDNA3.1-2B-miniSOG-flag.Under electron microscopy it was observed that 2B-miniS0G protein was located in the mitochondria,and a large number of vesicular structures appeared in the cytoplasm.Both autophagosomes and autophagic lysosomes can be observed.Conclusions Non-structural protein 2B of HRV16 can induce autophagy.