传染性法氏囊病病毒VP4酵母双杂交诱饵载体的构建及互作宿主细胞蛋白的筛选

VP4蛋白(viral protein 4)是传染性法氏囊病病毒(infectious bursal disease virus,IBDV)编码的一种重要非结构蛋白,具有蛋白水解酶活性,剪切多聚蛋白pVP2-VP4-VP3释放出pVP2、VP4、VP3,对病毒蛋白成熟具有重要的作用。为了研究VP4蛋白与宿主细胞的相互作用,本研究将IBDV VP4基因克隆入pGBKT7,构建诱饵载体pGBGtVP4,自激活试验证明其无自激活活性,对酵母细胞没有毒性。运用酵母双杂交技术,从鸡胚成纤维细胞(CEF)文库中筛选VP4的互作蛋白,经初筛、测序和回转验证,获得22个候选互作宿主细胞蛋白,为深入研究IBDV VP4与宿主互作的效应和机制奠定了基础。

Construction of Bait Vector of Infectious Bursal Disease Virus VP4 and Screening of the Interacted Proteins of Host Cell Using Yeast Two-hybrid System

Viral protein 4 (VP4) is an important non-structural protein with proteolytic enzyme activity encoded by infectious bursal disease virus (IBDV) genome that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form viral proteins pVP2,VP4,VP3,which plays an important role in the process of viral proteins maturity.To study the interaction between VP4 and the host cell,VP4 full-length cDNA sequence was amplified and inserted into pGBKT7 vector to construct a bait vector (pGBGtVP4).The result of autoactivation assay showed that pGBGtVP4 vector had no autoactivation and virulence in Y2H gold yeast cell.After screening,sequencing and confirming interaction,22 kinds of potential interacted proteins were obtained from the expression library of chicken embryo fibroblasts (CEF) using the matchmaker gold yeast two-hybrid system.This study laid the foundation for further researches on the effect and mechanism of the interaction of IBDV VP4 and its host cell.