酵母双杂交筛选与CVB3 VP3相互作用的人心脏蛋白  

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

作　　者：张志勤[1] 赵颖洁[1] 夏燕华[1] 王静[1] 项国仕[1] 邹叶青[2] 黄孝天[1] 

机构地区：[1]南昌大学医学院微生物学教研室,南昌330006 [2]南昌大学第二附属医院,南昌330006

出　　处：《中国人兽共患病学报》2014年第10期1014-1019,共6页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81160196;No.30660010);江西省青年科学家培养项目(20133BCB23005);江西省研究生创新专项资金项目(YC2012-S020);江西省自然科学基金(20120BAB205042)联合资助~~

摘　　要：目的从人心脏cDNA文库中筛选与B3型柯萨奇病毒(coxsackievirus group B type 3,CVB3)结构蛋白VP3相互作用的蛋白,为进一步研究CVB3分子致病机制提供新的线索。方法构建酵母双杂交重组质粒(pGBKT7-VP3),转化至感受态酵母菌AH109,检测BD-cMyc-VP3融合蛋白自激活,应用酵母双杂交筛选与CVB3VP3相互作用的人心脏蛋白。对阳性候选克隆进行测序和同源性比对分析;α-半乳糖苷酶活性定量分析VP3与各阳性蛋白之间相互作用的强弱。结果诱饵质粒pGBKT7-VP3构建成功,检测到BD-cMyc-VP3融合蛋白在AH109中表达,且诱饵蛋白VP3在酵母中不存在自激活,从人心脏cDNA文库中筛选到10个与CVB3VP3相互作用的蛋白:真核翻译起始因子4A2、羟酰辅酶A脱氢酶三官能蛋白转录变体3、肌钙蛋白I3型、平滑肌蛋白3、线粒体乙醛脱氢酶2等。结论本研究成功应用酵母双杂交技术筛选出与CVB3VP3相互作用的10个蛋白。为研究CVB3引起心肌炎和心肌疾病的分子致病机制提供了一些新的线索。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. The α-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 by α-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.

关 键 词：酵母双杂交 CVB3 VP3 蛋白相互作用 

分 类 号：R373[医药卫生—病原生物学]