Abstract:

In order to construct human tumor necrosis factor TNF-α and glutathione-s- transferase (GST) fusion gene vector for prokaryotic expression. The open reading frame (ORF) of Human tumor necrosis factor TNF-α (TNF-α) gene was amplified by using specific primers and ligated to pMD-18T simple vector to generate pMD-18T-TNFα. Then it was released from the T simple plasmid by enzyme digestion and subsequently ligated to pGEX-4T-1, which had been digested with the same restriction enzyme, to create pGEX-4T-1-TNFα. To express GST- TNF-α fusion protein in E. coli, pGEX-4T-1-TNFα was transformed to E. coli strain BL21 (DE3) and the expression condition was optimized by adjusting tempreture, time and so on. SDS-PAGE result indicated that the 51 kDa fusion protein was expressed. It can be purified and used to study its biological function, mechanism and prepare monoclonal antibody anti- TNF-α.