RIP140重组腺病毒的构建及在乳鼠心肌细胞中的表达

摘要

Aim The limited transfection efficiency for plasmid in primary neonatal rat cardiomyocytes,which are terminal differentiated cells,and long foreign DNA (the RIP140 gene sequence are as long as 3.5 kb) cause us to choose a better system to study RIP140 gene expression in primary non-replicative cells. Methods Full-length of RIP140 was cloned into pAdTracker-CMV shuttle vector,and then recombined with virus backbone pAdEasy-1 vector in BJ5183 bac-teria.Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determina-tion,and then transfected into AD293 cells for amplifi-cation.Titers of virus particles were determined by Tis-sue Culture Infectious Dose 50 (TCID50 )method and cell vitality was analyzed by CCK-8 kit in cardiomyo-cytes.RIP140 gene was identified by Western blot. Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into AdEasyTM sys-tem.Virus titers of Ad-RIP140 and Ad-GFP were 1011.3 and 1011.7 PFU·mL-1 ,respectively.Cell vitali-ty was not affected when the Multiplicity of Infection (MOI)was lower than 200.Green fluorescent protein (GFP)and Western blot analysis showed RIP140 gene was remarkably increased in cardiomyocytes for 12h in-fection by Ad-RIP140 (P<0.05 ).Conclusion Re-combinant adenovirus containing RIP140 gene was suc-cessfully constructed and effectively expressed in car-diomyocytes.These will be helpful for further research on the function of RIP140 in cardiomyocytes.%目的：原代乳鼠心肌细胞为终末期的分化细胞，常规的质粒转染效率低下。受体相互作用蛋白140（receptor-in-teracting protein 140，RIP140）目的基因长达3.5kb，为研究RIP140蛋白在非增殖型的心肌细胞表达情况，需要寻找一种更好的过表达系统。方法 RIP140基因全长序列克隆入pAdTrack-CMV穿梭载体中，在BJ5183细菌中与腺病毒骨架载体pAdEasy-1进行同源重组。经酶切及测序验证的阳性重组克隆转染入AD293细胞中进行病毒的包装与扩增。采用TCID50法测定病毒滴度、CCK-8试剂盒分析腺病毒对心肌细胞活力的影响。采用Western blot鉴定RIP140蛋白在心肌细胞中的表达情况。结果测序分析结果表明RIP140的全长序列正确克隆到 AdEasyTM腺病毒系统中。Ad-RIP140、Ad-GFP 的病毒滴度分别为1011.3和1011.7 PFU · mL－1。腺病毒感染复数在200以下时，对心肌细胞活力无影响。绿色荧光及Western blot分析显示Ad-RIP140感染心肌细胞12 h，RIP140表达明显增加（P＜0.05）。结论成功构建了RIP140全长序列的腺病毒载体，并使其蛋白有效表达于心肌细胞中，这将有助于后续进行RIP140在心肌细胞中的作用的研究。