采用正交设计优化HPV16L1重组蛋白表达的实验研究

通过正交试验对构建的人乳头瘤病毒（HPV）16型L1蛋白的重组表达菌株pQE31—HPV16L1／M15（pREP4）进行表达条件的优化，以增加目的蛋白的表达量。实验选取影响蛋白表达的4个主要因素，分别为诱导时间、诱导剂浓度、细菌密度和诱导温度，采用四因素两水平正交试验，通过SDS-PAGE及Western blot测定重组表达蛋白，以Tanon凝胶成像系统分析目的蛋白含量作为检测条件优化效果的指标，所得数据采用SAS软件进行统计分析。实验结果表明在37℃、IPTG0．5mmol／L，菌密度OD600为1．0的条件下诱导4h所得蛋白量最大；经统计正交分析的最佳诱导条件为37℃、0．5mmol／LIPTG和菌密度OD600为1．0的条件下诱导7h。经实验验证，应用正交分析的最佳条件诱导所得目的蛋白量最大，但实际中通常采用诱导4h条件。实验证明正交试验可以用于基因工程菌株蛋白表达条件优化过程，提高工作效率。

Optimization of HPV16L1 Recombinant Protein Expression Adopting Orthogonal Design

In order to increase expression of target protein, the expression conditions of a constructed in the lab pQE31-HPV16L1 recombinant expression strain were optimized through orthogonal experiment. The experiment chose and adopted four main influential factors to the protein expression for the orthogonal experiment at two levels, they were inducing time, concentration of inducer, density of bacteria, and inducing temperature respectively. SDS-PAGE and Western bolting were used to test the recombinant protein expression, and took protein content analyzed by Tanon gel imaging system as the effect indices of the optimization of testing conditions; and the obtained data were analyzed statistically adopting SAS software. The results showed that under 37 ℃ , IPTG 0.5 mmol/L, bacterial density OD600 at 0.1 induced for 4 hours the protein quantity was the largest; however according to statistic orthogonal analysis the optimized induciug conditions were 37℃ , IPTG 0. 5 mmol/L, bacterial density OD600 at 0.1 induced for 7 hours. It was proved through experiment that the optimized condition of induction to obtain protein quantity applying orthogonal analysis was the largest, yet practically and generally 4 hours was used for the inducing conditions. Therefore, it was proved that orthogonal experiment could be used in the protein expression condition optimization process for genetic engineered strain in order to promote the working efficiency.