Objective: To explore the optimal conditions for prokaryotic expression of melanoma-associated antigen D4 (MAGE-D4) gene engineering bacteria,and to enhance the production of target protein. Methods: MAGE-D4 recombinant plasmid was induced by different host bacteria (DH5α,TB1,Rosetta),starting time of induction (recombinant bacteria were induced after cultured for 0.5-3 hours),IPTG concentration (0.1-0.9 mmol/L) and induction time (1-8 hours).The expression and solubility of the target protein were analyzed by SDS-PAGE electrophoresis and Image J software scanning.MAGE-D4 antibody in serum of 35 patients with hepatocellular carcinoma was detected by indirect ELISA to identify the immune activity of MAGE-D4 fusion protein. Results: The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3).0.9 mmol/L IPTG was added when the OD 600 reached 0.8 and induced at 37 ℃ for 7 hours,which could effectively increase the expression of the target protein.The soluble MAGE-D4 fusion protein accounted for about 25.5% of the total cellular proteins.The positive rate of MAGE-D4 antibody in serum of hepatocellular carcinoma was 28.57%(10/35),The average titer of MAGE-D4 antibody in serum of patients was significantly higher than that in normal( P <0.05). Conclusion: In this study,the prokaryotic expression conditions of MAGE-D4 fusion protein were optimized,and the soluble recombinant protein with immunologic activity was obtained.
Journal of Guangxi Medical University
melanoma-associated antigen D4 (MAGE-D4)