CAJ | 학술논문

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CVB3 VP3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 V P3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CVB3 VP3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CVB3 VP3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。
목적：종인심장cDNA문고중사선여B3형가살기병독（coxsackievirus group B type 3，CVB3）결구단백VP3상호작용적단백，위진일보연구CVB3분자치병궤제제공신적선색。방법구건효모쌍잡교중조질립（pGBKT7-VP3），전화지감수태효모균AH109，검측BD-cMyc-VP3융합단백자격활，응용효모쌍잡교사선여CVB3 VP3상호작용적인심장단백。대양성후선극륭진행측서화동원성비대분석；α-반유당감매활성정량분석 V P3여각양성단백지간상호작용적강약。결과유이질립pGBKT7-VP3구건성공，검측도BD-cMyc-VP3융합단백재AH109중표체，차유이단백VP3재효모중불존재자격활，종인심장cDNA문고중사선도10개여CVB3 VP3상호작용적단백：진핵번역기시인자4A2、간선보매A탈경매삼관능단백전록변체3、기개단백I3형、평활기단백3、선립체을철탈경매2등。결론본연구성공응용효모쌍잡교기술사선출여CVB3 VP3상호작용적10개단백。위연구CVB3인기심기염화심기질병적분자치병궤제제공료일사신적선색。
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 .