拟除虫菊酯类农药降解酯酶EstA融合蛋白表达质粒的构建和诱导条件优化

Construction of Fusion Protein Expression Vector of Pyrethroid Pesticides-degrading Esterase EstA and Optimization of Inducible Expression Conditions

以前期克隆得到的拟除虫菊酯类农药降解基因estA(GenBank:DQ906143)为出发基因,构建表达系统,获得可溶性融合蛋白,并通过优化诱导表达条件,获得具有实际应用价值的相关应用参数。结果表明,成功构建了重组表达载体pMal-c2X-estA并转化至宿主菌BL21(DE3),获得酯酶estA基因诱导表达系统;通过SDS-PAGE凝胶电泳、以α-乙酸萘酯为底物的酶活性检测及Western blot方法,确认外源蛋白EstA在大肠杆菌宿主菌中成功表达并具有酯酶活性。重组基因工程菌可溶性原核表达诱导条件的优化试验表明,其最佳诱导表达条件是:Ca2+浓度1.0mmol/L、诱导初始OD600值0.7、诱导剂IPTG浓度0.1mmol/L、诱导初始pH值7、诱导温度26℃、诱导时间17.5h,优化后的酯酶活性(92.03U/mg)较优化前(44.64U/mg)提高了1倍多。

The aim of this research was to construct an expression system by ligating the esterase gene estA （GenBank： DQ906143） cloned before, which could degrade pyrethroid pesticides, into the vector pMal-c2X to attain soluble protein; in addition, the expression conditions were optimized to achieve the related application parameters, which had great significance in application. The results showed that the recombinant expression vector pMal-c2X-estA was constructed and transformed into the host strain BL21（DE3）,and the inducible expression system of estA gene was obtained. Through the SDS-PAGE analysis, the enzyme activity assay using the α-naphthyl acetate as substrate and Western blot, it was confirmed that the target protein EstA was expressed in Escherichia coli and had esterase activity. Optimization of soluble prokaryotic expression conditions for the genetic engineering strain indicated that the optimal expression conditions were as follows：the concentration of Ca2＋ was 1.0 mmol/L,the initial OD600 value was 0.7,the concentration of IPTG was 0.1 mmol/L, the initial pH value was 7, the induction temperature was 26 ℃ ,and the induction time was 17.5 h. Under the optimal conditions,approximately double ex- pressed protein could be obtained compared with that before （the esterase activity was 44.64 U/mg before and 92.03 U/rag after）.