天蚕素AD和BuforinⅡ表达载体的改良和融合蛋白表达条件优化

Improvement of Cecropin AD and Buforin Ⅱ Expression Vector and Optimization for Fusion Protein Expression Conditions

摘要: 为提高重组大肠杆菌表达天蚕素AD和BuforinⅡ融合蛋白的表达效率, 研究表达菌株、质粒稳定性和诱导条件对天蚕素AD和BuforinⅡ融合蛋白表达的影响。通过SDS-PAGE分析目的蛋白占全菌总蛋白的含量, 确定大肠杆菌BER2566为表达菌株;利用基因工程技术, 构建卡那霉素抗性的表达质粒pET (K) -Trx-CADBuforinII, 提高质粒稳定性和蛋白表达效率;优化了IPTG浓度、诱导时机和诱导时间等诱导条件, 确定菌体浓度为OD600=0.8时, IPTG浓度为0.8mmol·L-1, 诱导时间为5h, 目的蛋白表达量最高, 可达到全菌总蛋白含量的50%以上。

Abstract: Effects of expression strains, plasmid stability and inducing conditions were studied on improving the expression efficiency of Cecropin AD and Buforin Ⅱ fusion protein in recombinant Escherichia coli.According to the result of SDS-PAGE, E.coli BER2566was accepted as expression strain.Plasmid pET (K) -Trx-CAD-Buforin Ⅱ with kanamycin resistant gene was constructed to improve the plasmid stability and protein expression.Inducing conditions including IPTG concentration, induction time and inducing time length were optimized.With the optimal conditions, the highest expression level of fusion protein was obtained in recombinant E.coli at the level of OD600 0.8and final IPTG concentration of 0.8mmol·L-1 for 5hinducing, which was more than 50%of the total protein in E.coli.