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[目的]为了提高植原体膜蛋白Imp基因在E.coliBL21(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.coliBL21(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GeneTools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℃,pH7.0,装液量20%,振荡速度200r/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6h。[结论]在最佳条件下Imp表达量达到70.98mg/L,确定了Imp融合蛋白在大肠杆菌的优化表达条件。
[Objective] The aim of this study was to optimize the expression conditions of Imp gene in E. coli BL21 (DE3) in order to improve the expression level of Imp gene. [Method] The effects of different culture conditions on E. coli BL21 (DE3) -pET-28a (+) - Imp were studied by orthogonal design. On the basis of obtaining the best culture conditions, the effects of different induction conditions on the expression of Imp protein were investigated. The expression level of fusion protein Imp was analyzed by SDS-PAGE and GeneTools gel analysis software. [Result] Optimization of expression conditions showed that the optimal culture conditions were as follows: temperature 37 ℃, pH7.0, liquid volume 20%, shaking speed 200r / min. The best induction conditions were as follows: temperature 37 ℃, initial OD600≈1.5, final IPTG concentration 0.1mmol / L, induction culture time 6h. [Conclusion] The optimal expression of Imp fusion protein in Escherichia coli was determined under the optimum conditions when the expression of Imp reached 70.98 mg / L.