Construction of small hair RNA eukaryotic expression vectors targeting TGF-?_1 gene and its inhibitory effects on TGF-?_1 gene / 重庆医科大学学报

ABSTRACT

Objective:To construct short hairpin RNA(shRNA)eukaryotic expression vector targetinghuman TGF-?1 gene,and evaluate its inhibitory effects on TGF-?1 gene in endothelial cell lin(eECV304)of human umbilical veins in order to select a high efficacy vector for subsequent RNAi study.Methods:The oligonucleotides targeting TGF-?1 mRNA were designed and synthesized according to bioinformatics methods.The annealed duplex strand were cloned into PGenesil-1 plasmid.Two recombinant plasmids(pT11 and pT12) were verified by enzyme digestion and sequencing,and transfected into endothelial cell line of human umbilical veins.Vector with scramble sequence was set as control.The mRNA expression was measured by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and the protein was measured by ELISA.Results:The shRNA expressing vectors targeting TGF-?1 gene were confirmed by SalI enzyme digestion and sequencing.The transfection rate of pT11 and pT12 in ECV304 were 38.2%and 40.1%,respectively.The inhibitory rate of TGF-?1 mRNA were 58.1%and 60.0%,and the inhibitory rate of TGF-?1 protein were 38.9%and 44.3%.Conclusions:The successfully constructed shRNA eukaryotic expression vectors specific for human TGF-?1 gene have significant inhibitory effects on the expression of TGF-?1 mRNA and protein in endothelial cell line of human umbilical veins,and the pT12 is more effective.