RIP140重组腺病毒的构建及在乳鼠心肌细胞中的表达
|
摘要
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-interacting 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p Ad Track-CMV穿梭载体中,在BJ5183细菌中与腺病毒骨架载体p Ad Easy~(-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Ad Easy TM腺病毒系统中。AdRIP140、Ad-GFP的病毒滴度分别为1011.3和1011.7PFU·m L~(-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。
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 p Ad Tracker-CMV shuttle vector,and then recombined with virus backbone p Ad Easy-1 vector in BJ5183 bacteria. Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determination,and then transfected into AD293 cells for amplification. Titers of virus particles were determined by Tissue Culture Infectious Dose 50( TCID_(50)) method and cell vitality was analyzed by CCK-8 kit in cardiomyocytes. RIP140 gene was identified by Western blot.Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into Ad Easy TM system. Virus titers of Ad-RIP140 and Ad-GFP were1011. 3and 1011. 7PFU·m L~(-1),respectively. Cell vitality 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 12 h infection by Ad-RIP140( P < 0. 05). Conclusion Recombinant adenovirus containing RIP140 gene was successfully constructed and effectively expressed in cardiomyocytes. These will be helpful for further research on the function of RIP140 in cardiomyocytes.
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 p Ad Tracker-CMV shuttle vector,and then recombined with virus backbone p Ad Easy-1 vector in BJ5183 bacteria. Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determination,and then transfected into AD293 cells for amplification. Titers of virus particles were determined by Tissue Culture Infectious Dose 50( TCID_(50)) method and cell vitality was analyzed by CCK-8 kit in cardiomyocytes. RIP140 gene was identified by Western blot.Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into Ad Easy TM system. Virus titers of Ad-RIP140 and Ad-GFP were1011. 3and 1011. 7PFU·m L~(-1),respectively. Cell vitality 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 12 h infection by Ad-RIP140( P < 0. 05). Conclusion Recombinant adenovirus containing RIP140 gene was successfully constructed and effectively expressed in cardiomyocytes. These will be helpful for further research on the function of RIP140 in cardiomyocytes.
引文
[1]Docquier A,Augereau P,Lapierre M,et al.The RIP140 gene is a transcriptional target of E2F1[J].Plo S One,2012,7(5):e35839.
[2]Fritah A,Steel J H,Parker N,et al.Absence of RIP140 reveals a pathway regulating glut4-dependent glucose uptake in oxidative skeletal muscle through UCP1-mediated activation of AMPK[J].Plo S One,2012,7(2):e32520.
[3]Fritah A,Steel J H,Nichol D,et al.Elevated expression of the metabolic regulator receptor-interacting protein 140 results in cardiac hypertrophy and impaired cardiac function[J].Cardiovascular Res,2010,86(3):443-51.
[4]陈艳芳,刘培庆.RIP140/PGC-1a在Ang II调节心肌能量代谢中的作用研究[J].中国药理学通报,2015,31(2):194-8.[4]Chen Y F,Liu P Q.The role of RIP140 and PGC-1αin the Angiotensin II mediated energy metabolism in cardiomyocytes[J].Chin Pharmacol Bull,2015,31(2):194-8.
[5]He T C,Zhou S,da Costa L T,et al.A simplified system for generating recombinant adenoviruses[J].Proc Natl Acad Sci USA,1998,95(5):2509-14.
[6]Luo J,Deng Z L,Luo X,et al.A protocol for rapid generation of recombinant adenoviruses using the Ad Easy system[J].Nat Protoc,2007,2(5):1236-47.
[7]王爽,许坚吉,刘晓霓,陈德喜.人ASPP2重组腺病毒质量控制研究[J].中国药理学通报,2016,32(6):881-5.[7]Wang S,Xu J J,Liu X N,Chen D X.Quality control of human ASPP2 recombinant adenovirus[J].Chin Pharmacol Bull,2016,32(6):881-5.
[2]Fritah A,Steel J H,Parker N,et al.Absence of RIP140 reveals a pathway regulating glut4-dependent glucose uptake in oxidative skeletal muscle through UCP1-mediated activation of AMPK[J].Plo S One,2012,7(2):e32520.
[3]Fritah A,Steel J H,Nichol D,et al.Elevated expression of the metabolic regulator receptor-interacting protein 140 results in cardiac hypertrophy and impaired cardiac function[J].Cardiovascular Res,2010,86(3):443-51.
[4]陈艳芳,刘培庆.RIP140/PGC-1a在Ang II调节心肌能量代谢中的作用研究[J].中国药理学通报,2015,31(2):194-8.[4]Chen Y F,Liu P Q.The role of RIP140 and PGC-1αin the Angiotensin II mediated energy metabolism in cardiomyocytes[J].Chin Pharmacol Bull,2015,31(2):194-8.
[5]He T C,Zhou S,da Costa L T,et al.A simplified system for generating recombinant adenoviruses[J].Proc Natl Acad Sci USA,1998,95(5):2509-14.
[6]Luo J,Deng Z L,Luo X,et al.A protocol for rapid generation of recombinant adenoviruses using the Ad Easy system[J].Nat Protoc,2007,2(5):1236-47.
[7]王爽,许坚吉,刘晓霓,陈德喜.人ASPP2重组腺病毒质量控制研究[J].中国药理学通报,2016,32(6):881-5.[7]Wang S,Xu J J,Liu X N,Chen D X.Quality control of human ASPP2 recombinant adenovirus[J].Chin Pharmacol Bull,2016,32(6):881-5.