SA11株轮状病毒VP3基因的克隆表达及致病机制的初步研究  

Cloning and expression and preliminary study of the pathogenic mechanism of SA11 rotavirus VP3 gene

作　　者：李明[1,2] 何海洋[2] 徐广振[1] 尹怡[1] 薛毅[1] 吴玉章[2] 李晋涛[2] 

机构地区：[1]第三军医大学学员旅,重庆400038 [2]第三军医大学全军免疫学研究所

出　　处：《免疫学杂志》2011年第6期466-469,481,共5页Immunological Journal

基　　金：国家自然基金资助项目(30400402;30571708);重庆市科技攻关计划项目(CSTC2009AB5197)

摘　　要：目的克隆、真核表达SA11株轮状病毒VP3基因,对其致病机制进行了初步研究。方法用RT-PCR从SA11株总RNA中扩增出VP3基因,并克隆到真核表达载体pEGFP-C1上,构建重组表达载体pEGFP-C1/VP3,用荧光显微镜及Western blot检测VP3基因在真核细胞(293T细胞)的表达情况。以重组质粒pEGFP-C1/Rb94为对照,用流式、荧光显微镜、Western blot观察VP3蛋白对GFP基因表达的抑制作用。结果在293T细胞中检测到VP3基因的表达;pEGFP-C1/VP3组的流式荧光强度、Western blot的蛋白条带大小均明显弱于pEGFP-C1/Rb94对照组。结论成功构建了pEGFP-C1/VP3真核穿梭表达载体,在真核细胞中有效表达,实验结果提示VP3蛋白可能对宿主细胞大分子蛋白的表达有抑制作用,可能是轮状病毒的一种新的致病机制。To explore the relationship between SA11VP3 and the host cell,as well as study its potential molecular mechanism,the co-expression vector of VP3 of SA11 with EGFP was constructed.RT-PCR was preformed to amplify VP3 gene fragments from the reverse transcription product of RV genome RNA.The VP3 gene fragments were harvested and inserted into pEGFP-C1 to construct the pEGFP-C1/VP3 expression vector.And the expression in 293T cell was observed under a fluorescence microscope and analyzed by Western blotting.As a control with pEGFP-C1/Rb94 plasmid,the inhibition of VP3 protein to GFP gene was detected by Flow cytometry,fluorescence microscope and Western blotting.Western blotting demonstrated that pEGFP-C1/VP3 could express the VP3 protein in 293T cell.Fluorescence intensity of pEGFP-C1/VP3 group was significant weaker than that of pEGFP-C1/Rb94 group.In conclusion,a recombinant eukaryotic co-expression vector of pEGFP-C1/VP3 was successfully constructed,which is effectively expressed in eukaryotic cells.VP3 protein may inhibit the expression of host cell gene.All the results provide a possible pathogenic mechanism of rotavirus VP3.

关 键 词：轮状病毒 VP3基因 真核表达 致病机制 

分 类 号：R373.2[医药卫生—病原生物学] Q786[医药卫生—基础医学]