关键词: 人可溶性肿瘤病坏死因子受体1, 真核表达, 瞬时转染, 细胞毒性作用

Abstract: Objective To construct an eukaryotic expression vector of human sTNFR1 and investigate its inhibitory effects of the bioactivity of TNFα. 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 TNFα.Results The exactitude of recombinant vector pCDNA3.1(-)-sTNFR1 was identified by digestion and DNA sequencing analysis, After being trancfected pcDNA3.1(-)-sTNFR1, QSG7701 had higer expression level of sTNFR1mRNA than the pcDNA3.1(-) trancfected control; After the recombinant plasmid pcDNA3.1(-)-sTNFR1 was transfected into QSG7701 cell transiently. The cytotoxic effect of TNFα was inhibited to the extent of 64.8% when its concentration was 100ng/mL. Conclusion We constructed the eukaryotic expression vector containing Human sTNFR1 gene successfully, the cytotoxicity of TNFαcan be inhibited in pcDNA3.1(-)- sTNFR1/QSG7701 cells.

Key words: human sTNFR1, eukaryotic expression, transiently transfection, cytotoxicity