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

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(英文)

作　　者：柴化建[1] 赵海泉[1] 张丽君 罗焕亮[3] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036 [2]深圳市职业技术学院,广东深圳518055 [3]深圳出入境检验检疫局,广东深圳518045

出　　处：《Agricultural Science & Technology》2012年第3期520-524,557,共6页农业科学与技术（英文版）

基　　金：Supported by Science and Technology Project of Shenzhen City (JC200903180710A)~~

摘　　要：[Objective] This study aimed to increase the expression level of immun- odominant membrane protein gene （Imp） of phytoplasmas in E. coil BL21 （DE3）. [Method] On the basis of orthogonal experiment, effects of different culture conditions on recombinant bacteria E. coil BL21-pET-28a（＋）-Imp were investigated. Based on the obtained optimal culture condition, effects of different induction conditions on the ex- pression level of Imp protein were explored. The expression level of Imp fusion pro- tein was analyzed by using SDS-PAGE and Gene Tools software. [Result] The re- sults showed that the optimal conditions for culture were at 37℃, pH 7.0, with liq- uid volume of 20% and oscillation speed of 200 r/min, 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 achieved 70.98 mg/L under the optimal conditions. Optimized conditions for expression of Imp fusion protein in E. coil were determined.[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。

关 键 词：PHYTOPLASMA Imp gene Expression condition Orthogonal experimental design 

分 类 号：S432.4[农业科学—植物病理学]