Construction of the Eukaryotic Expression Vectors and the microRNA Expression Plasmids of Human Fas and TNFR1 Gene and Their Biological Effects in vitro
10.3870/j.issn.1672-0741.2010.01.013
- VernacularTitle:人重型肝炎相关基因Fas、TNFR1真核表达载体和microRNA表达载体的构建及其体外干预效应的初步研究
- Author:
Sui GAO
;
Dong XI
;
Jianwen GUO
;
Weiming YAN
;
Xiaoping LUO
;
Qin NING
- Publication Type:Journal Article
- Keywords:
Fas;
TNFR1;
microRNA;
apoptosis;
fulminant hepatitis
- From:
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
2010;39(1):50-54
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression plasmids of human Fas and TNFR1 gene(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and microRNA(miRNA)expression plasmid of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA,and to investigate their inhibitory effects in vitro.Methods The eukaryotic expression plasmids of human Fas and TNFR1 gene were constructed(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and have been shown successfully to express hFas and hTNFR1 protein.miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA complimentary to the sequence responsible for hFas and hTNFR1 respective were constructed,meanwhile irrelevant miRNA plasmid was used as a control.By respective co-transfection of p-hFasmiRNA and pcDNA3.0-hFas,p-hTNFR1 miRNA and pcDNA3.0-hTNFR1 expression construct into 293T cells,the inhibition of hFas and hTNFR1 expression was analyzed by real-time PCR and Western blot.Results The experiments showed the significant inhibitory effect of p-hFasmiRNA on hFas and p-hTNFR1miRNA on hTNFR1 expression at 48 h post-transfection both at RNA level and protein level as well in 293T cell lines with the inhibitory efficiency being as high as 87% for hFas and 80% for hTNFR1,respectively.Conclusion The p-hFasmiRNA and p-hTNFR1miRNA were constructed successfully,and it was verified that they could specifically inhibit the hFas and hTNFR1 expression at the cellular level.
