ABSTRACT
Objective To construct a human tumor necrosis factor (hTNF-a) plasmid and identify it to optimize the fermentation conditions of hTNF-α protein so as to achieve high expression in Escherichia coli.Methods The gene of hTNF-a was cloned into pET24a vector to obtain the pET24a-hTNF-a expression plasmid that was transformed into Escherichia coli BL21(DE3),and the expression conditions of BL21 (DE3) were optimized.Results The plasmid of pET24a-hTNF-α was successfully constructed and identified by PCR and digestion,which was consistent with the target fragment hTNF-α.The plasmid was transformed into Escherichia coli BL21(DE3),the best induced expression conditions of Escherichia coli BL21 (DE3) were as follows:M9+LB medium,37℃,0.5 mmol/L IPTG,pH =7.5,and induction time was 5 h.The results showed that dry weight of the cells and the rate of TNF were increased by 2.56 times and 3.68 times,respectively,and the expression rate of hTNF-α was increased by 3.49 times from 9.38％ to 32.74％.Conclusion The optimal conditions for the expression of plasmid pET24a-hTNF-α in Escherichia coli were determined.