植原体免疫主导膜蛋白Imp基因在大肠杆菌中表达条件优化

目的]为提高植原体膜蛋白Imp基因在E.coli BL21(DE3)中的表达量,优化Imp基因的原核表达条件。方法]通过设计正交试验,考察不同的培养条件对工程菌E.coli BL21(DE3)-pET-28a(+)-Imp的影响。在获得最佳培养条件的基础上考察不同诱导条件对Imp蛋白表达量的影响。利用SDS-PAGE和Gene Tools凝胶分析软件分析融合蛋白Imp的表达量。结果]表达条件优化结果表明,最佳培养条件为:温度37℃,pH 7.0,装液量20%,振荡速度200 r/min;最佳诱导条件为:温度37℃,起始OD600≈1.5,IPTG终浓度0.1mmol/L,诱导培养时间6 h。结论]在最佳条件下Imp表达量达70.98 mg/L,确定了Imp融合蛋白在大肠杆菌的优化表达条件。

Optimization of Expression Conditions for Immunodominant Membrane Protein Gene of Phytoplasmas in E.coli

Objective] This study aimed to increase the expression level of immunodominant membrane protein gene(Imp) of phytoplasmas in E.coli BL21(DE3).Method] On the basis of orthogonal experiment,effects of different culture conditions on recombinant bacteria E.coli BL21-pET-28a(+)-Imp were explored.Based on the obtained optimal culture condition,effects of different induction conditions on the expression level of Imp protein were explored.The expression level of Imp fusion protein was analyzed by using SDS-PAGE and Gene Tools software.Result] The results showed that the optimal conditions for culture were at 37 ℃,pH 7.0,with liquid volume of 20% and oscillation speed of 200 r/min;the optimal conditions for induction were at 37 ℃ for 6 h,with initial OD600 of about 1.5 and IPTG final concentration of 0.1 mmol/L.Conclusion] The expression level of Imp had achieved 70.98 mg/L under the optimal conditions.Optimized conditions for expression of Imp fusion protein in E.coli were determined.