基因工程菌诱导表达苏云金芽胞杆菌Vip3Aa蛋白的条件优化

摘要

Vegetative insecticidal protein (Vip) is secreted by Bacillus thuringiensis (Bt) during the period from vegetative growth to logarithmic metaphase. To obtain efficient expression condition of Vip3Aa protein, 3 main factors (concentration of isopropyl- β- d- thiogalactoside(IPTG), inducing time and inducing temperature) were screened for the optimization inducing condition of recombinant Escherichia coli BL21-pCzn1-Vip in producing the Vip3Aa by orthogonal experiment. The highest expression level of target protein could be attained with the condition of 200μg/mL IPTG inducing for 8 h at 21℃. The data were analyzed by direct calculation and one-way ANOVA method. R value (resulting from direct calculation) of inducing temperature was the highest among the 3 factors, followed by the inducing time and the concentration of IPTG, wihch indicated that the inducing temperature was the most important factor to affect expression level of target protein. P value from one-way ANOVA of inducing temperature and inducing time was lower than 0.01 and 0.05, respectively, and the P value of the other factor was higher than 0.05, which were similar as the results of direct calculation. The amount of the expressed target protein at the optimal inducing condition was up to 27.6% of total protein, which was 2.3 times of the production before optimization. It can provide the basis for the Vip3Aa manufacture, in-depth theoretical research and practical application.%营养期杀虫蛋白(vegetative insecticidal protein, Vip)是在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)营养生长至对数中期到稳定期期间产生的一类蛋白。为获得苏云金芽胞杆菌Vip3Aa蛋白的高效表达条件，本研究利用正交试验综合考察了大肠杆菌(Escherichia coli)重组基因工程菌BL21-pCzn1-Vip诱导过程中诱导剂浓度、诱导时间和诱导温度对Vip3Aa蛋白表达量的影响。结果表明，该工程菌目的蛋白的最优诱导表达条件：诱导温度21℃、诱导时间8 h、诱导剂(异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside, IPTG))浓度为200μg/mL；直观分析表明，影响目的蛋白表达量的最主要因素为诱导温度，其次为诱导时间，诱导剂浓度影响最小；方差分析结果表明，诱导温度的P值＜0.01、诱导时间的P值＜0.05、诱导剂浓度的P值＞0.05，与直观分析结果相一致；利用优化后诱导表达条件进行培养，测得目的蛋白最高表达量可占总蛋白的27.6%，相当于常规诱导方法的2.3倍。本研究为大量制备高纯度Vip3Aa蛋白及其深入的理论研究和实践应用提供了基础资料。