正交试验设计优化重组蛋白溶细胞毒素的表达

Optimizing the inducing conditions of expression of recombinant hemolysin in E. coli BL21/DE3 by orthogonal design

目的:对创伤弧菌溶细胞毒素融合蛋白表达条件进行优化,从而获得高效稳定的溶细胞毒素(Vibrio vulnificus Hemolysin,VVH)原核表达系统。方法:质粒pGEX-4T-1-vvh转化E.coli BL21/DE3后,针对诱导过程中对工程菌表达蛋白产生影响的4个因素:诱导时间、诱导温度、诱导剂浓度以及诱导时摇菌的转数,运用统计学软件进行正交实验设计,对结果进行直观分析及方差分析。并对实验得出的最佳搭配诱导条件进行验证。结果:直观分析表明诱导时间的R值最大,其次为摇菌转速和诱导剂IPTG浓度,诱导温度的R值最小;方差分析表明诱导时间的P<0.05,而转速、温度及IPTG浓度的P均>0.05。结论:诱导时间为主要因素(其为4h,表达水平最高),其次为摇菌转速、诱导剂IPTG浓度和诱导温度。本实验的最佳搭配诱导条件为摇菌转数为250r/min、诱导时间4h、诱导温度30℃、诱导剂IPTG浓度1mmol/L。在此诱导条件下,溶细胞毒素的表达量达到了55.31%。

Objective： To optimize the expression of recombinant hemolysin of Vibrio vulnificus in E.coli BL21/DE3 induced by IPTG.Methods： The plasmid of pGEX-4T-1-vvh/E.coli BL21 was constructed by our laboratory.Four different factors,including time,temperature,agitation speed and concentration of IPTG,were studied by orthogonal experimental design.The data were analyzed by direct calculation and ANOVA method.Results： The R value of time resulting from direct calculation was the highest among four factors.The P value of time was lower than 0.05,while P value of the other three factors were higher than 0.05.Conclusions： The expression level is remarkably affected by time.The expression level of VVH protein is the highest when inducing time is 4 hours.Therefore we have established a method for high-level expression of VVH,specifically,four hours-inducing time,30 ℃ inducing temperature,the 1 mmol/L concentration of IPTG and the agitation rate in 250 rpm.