摘要
目的以CVB3非结构蛋白3A为诱饵,从人心脏cDNA文库中筛选与其相互作用的宿主蛋白,对阳性cDNA克隆进行初步分析和鉴定。方法构建pGBKT7-3A重组质粒,Western Blot检测3A融合蛋白的表达;利用酵母双杂交技术筛选人心脏cDNA文库,经PCR扩增cDNA插入片段和Alu I酶切等实验将阳性克隆归类,并通过测序、相似性比对分析和回返配合实验去除假阳性蛋白。结果成功构建诱饵质粒pGBKT7-3A,Western Blot结果表明,pGBKT7-3A重组质粒在酵母菌中成功表达3A融合蛋白;经双杂交筛选和回返配合实验,获得了7个相互作用的蛋白,分别为:兰尼碱受体2(Ryanodine Receptor 2,RyR2),信号肽酶2(Signal peptidase complex subunit 2,SPCS2),跨膜蛋白emp24(Transmembrane emp24protein transport domain containing 4,TMED4),细胞色素c氧化酶亚基I(Cytochrome c oxidase subunit I,COX1),细胞色素c氧化酶亚基III(Cytochrome c oxidase subunit III,COX3),肌球蛋白重链(Myosin heavy chain,MYH7)和琥珀酸脱氢酶亚基D(Succinate dehydrogenase complex subunit D,SDHD)。结论应用酵母双杂交技术,以CVB3 3A为诱饵蛋白,从人心脏cDNA文库中获得7种不同的阳性基因:RyR2、SPCS2、TMED4、COX1、COX3、MYH7和SDHD,在分子水平上探索3A蛋白的新功能。
We used CVB3 3A as a bait to screen human heart interacting proteins by yeast two-hybrid system and ana- lyzed candidate positive colonies in the study. The bait plasmid pGBKT7-3A was constructed, and 3A fusion protein in AH109 yeast cells was detected. After screening proteins interacted with CVB3 3A from a human heart cDNA library, PCR amplifica- tion of cDNA inserts and Alu I cutting were used to sort positive colonies to eliminate duplicates; positive genes were se- quenced, similarly analyzed, and confirmed by yeast mating. The results showed that the 3A fusion protein was successfully expressed in AH109. Seven binding proteins of CVB3 3A were obtained from human heart cDNA library： Ryanodine Receptor 2 （RyR2）, Signal peptidase complex subunit 2 （SPCS2）, Transmembrane emp24 protein transport domain containing 4 （TMED4）, Cytochrome c oxidase subunit I （COX1）, Cytochrome c oxidase subunit III （COX3）, Myosin heavy chain（MYH7）, and Succinate de- hydrogenase complex subunit D （SDHD）. This study would contribute to exploration of the 3A function on molecular level.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第2期146-149,共4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年度计划资助(20133BCB23005)
江西省研究生创新专项资金项目(No.YC2012-S020)联合资助~~
