Screening CVB3 VP3 interacting proteins from cDNA library of human heart by yeast two hybridization

VernacularTitle:酵母双杂交筛选与CVB3 VP3相互作用的人心脏蛋白
Author: Zhiqin ZHANG ; Yingjie ZHAO ; Yanhua XIA ; Jing WANG ; Guoshi XIANG ; Yeqing ZOU ; Xiaotian HUANG
Publication Type:Journal Article
Keywords: yeast two hybridization; CVB3; VP3; protein-protein interaction
From: Chinese Journal of Zoonoses 2014;(10):1014-1019
CountryChina
Language:Chinese
Abstract: To screen interaction proteins of CVB3 VP3 from cDNA library of human heart ,yeast two hybridization was conducted in this study .The bait plasmid pGBKT7-VP3 was constructed ,VP3 fusion protein and its self-activation in AH109 yeast cells was then detected .The positive clones were confirmed by PCR amplification of cDNA inserts ,Alu I digesting ,DNA sequencing ,and Blasting were used to sort positive colonies to eliminate duplicates .Positive clones were confirmed by one-to-one yeast two hybridization ,and them were sequenced and analyzed for homology .Theα-galactosidase assay was performed to detect the interaction strength .Totally ,10 positive proteins interacting with VP3 of CVB3 were obtained by homology analy-sis,namely,EIF4A2,HADHB,GAPDH,ASPG,ACTA1,TNNI3,CKM,LMOD3,ERGIC1,and ALDH2.The strength of interactions between VP3 and 10 candidate proteins were proved byα-galactosidase assay .This study will contribute to explore the CVB3 VP3 function on molecular level and provides some new clues to explain the pathogenic mechanism of myo-carditis and cardiomyopathy .