Construction of eukaryotic recombinant sTNFR1 plasmid and its inhibitory effects on TNF-? cytotoxicity in vitro / 基础医学与临床

ABSTRACT

Objective To construct an eukaryotic expression vector of human sTNFR1 and to investigate its inhibitory effects to the bioactivity of TNF-?.Methods The total RNA was extracted from HeLa cells and used as a template to amplify human sTNFR1 gene by reverse transcription polymerase chain reaction(RT-PCR).The PCR products were cloned into T vector and sub-cloned into vector pcDNA3.1(-),an eukaryotic expression vector.The recombinant plasmid pcDNA3.1(-)-sTNFR1 was transfected into QSG7701 cells by using lipofectamine,RT-PCR was performed to detect the expression of sTNFR1,MTT was used to observe sTNFR1 gene 's inhibitory effect on TNF-?.Results QSG7701 has a higer expression level of sTNFR1 mRNA than pcDNA3.1(-) trancfected control.The cytotoxic effect of TNF-? was inhibited to the extent of 64.8% when its concentration was 100 ?g/L.Conclusion We constructed the eukaryotic expression vector containing human sTNFR1 gene and the cytotoxicity of TNF-? is inhibited in pcDNA3.1(-)-sTNFR1/QSG7701 cells.