中国药理学通报2016，Vol.32Issue(12)：1735-1740,6.DOI:10.3969/j.issn.1001-1978.2016.12.019
RIP140重组腺病毒的构建及在乳鼠心肌细胞中的表达
Construction of RIP140 recombinant adenovirus and its expression in neonatal rat cardiomyocytes
摘要
Abstract
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.关键词
腺病毒/同源重组/感染复数/细胞病理效应/半数组织感染剂量/心肌细胞Key words
adenovirus/homologous recombination/multiplicity of infection/cytopathic effect/tissue cul-ture infectious dose 50/cardiomyocytes引用本文复制引用
陈艳芳,王若伦,刘培庆..RIP140重组腺病毒的构建及在乳鼠心肌细胞中的表达[J].中国药理学通报,2016,32(12):1735-1740,6.基金项目
国家自然科学基金资助项目 （）
