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.
出处
《免疫学杂志》
CAS
CSCD
北大核心
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)
关键词
轮状病毒
VP3基因
真核表达
致病机制
Rotavirus
VP3 gene
Eukaryotic expression
Pathogenic mechanism