摘要
目的:对创伤弧菌金属蛋白酶融合蛋白表达条件进行优化,从而获得高效稳定的金属蛋白酶原核表达系统。方法:质粒pET-32a-vvp转化E.coliBL21/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:直观分析表明摇菌转数的R值最大,其次为诱导时间和诱导温度,诱导剂IPTG浓度的R值最小;方差分析表明摇菌转数的P值小于0.05,而时间因素、温度及IPTG浓度的P值均大于0.05。结论:摇菌转数为主要因素(其为250r/m时,表达水平最高),其次为诱导时间,而诱导温度和诱导剂IPTG浓度对实验结果影响不明显。本实验的最佳搭配诱导条件为摇菌转数250r/m、诱导时间4h、诱导温度37℃、诱导剂IPTG浓度1mmol/L。在此诱导条件下,金属蛋白酶的表达量高达40.67%。
Objective. To optimize the expression of recombinant metalloprotease of Vvibrio vulnificus in E. coliBL21/DE3 induced by IPTG. Method： The plasmid of pET-32a-vvp/E, coliBL21 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 agitation rate resulting from direct calculation was highest among four factors. The P value of agitation rate was lower than 0.05. The P value of the other factors were higher than 0.05. Conclusions. The expression level is remarkable affected by the agitation rate. The expression level of VVP protein is highest when agitation rate is 250 rpm. Therefore we have established a method for high-level expression of VVP-four hours-inducing time, 37℃-inducing temperature, 1mmol/L concentration of IPTG and agitation rate in 250rpm.
出处
《数理医药学杂志》
2008年第3期276-279,共4页
Journal of Mathematical Medicine
关键词
创伤弧菌
金属蛋白酶
正交设计
直观分析
方差分析
vibrio vulnificus
metalloprotease
orthogonal experimental design
direct analysis
analysis of variance（ANOVA）
