抗菌肽Plectasin原核表达条件的正交试验优化研究

Research on Optimization Expression Conditions of Plectasin Prokaryotic Expressions by the Orthogonal Experiment

为提高重组抗菌肽Plectasin的表达量,对构建的Plectasin重组表达载体p E-SUMO-PS的表达条件进行正交试验优化。根据Plectasin氨基酸序列合成了Plectasin基因,通过限制性内切酶Xba I和Bsa I插入表达载体p E-SUMO中,转化E.coli Origami(DE3),IPTG诱导表达。选取诱导时间,诱导浓度和诱导温度共3个主要外部因素。采用3因素3水平正交试验设计,通过SDS-PAGE测定重组表达蛋白。以标准SDS-PAGE图法分析目的融合蛋白含量,以单位上清体积内融合蛋白量作为检测指标,结果用SPSS13.0软件进行统计分析。实验成功构建了重组表达载体p E-SUMO-PS,且融合蛋白主要是以可溶性蛋白为主。在30℃,0.05 mmol/L IPTG的条件下诱导6 h所得融合蛋白量最大。影响抗菌肽Plectasin融合表达的因素的强度由高到低分别为诱导温度、诱导时间及诱导剂浓度。通过正交试验得出抗菌肽Plectasin原核表达的最佳诱导条件为30℃,6 h和0.05 mmol/L IPTG。这对于抗菌肽Plectasin的后续纯化及产业化具有重要参考价值。

The expression conditions of recombinant expression vector p E-SUMO-PS constructed in lab were optimized by the orthogonal experiment,so as to increase the expression of target protein. The gene encoding plectasin was designed based on the amino acid sequence of plectasin,inserted into the expression vector p E-SUMO through the restriction enzymes Xba I and Bsa I. Then the recombinant plasmid was transformed into E. coli Origami( DE3). The fusion protein was induced by IPTG. Three main influential factors to the expression of plectasin,which were inducing time,concentration of inducer and inducing temperature,were chosen and adopted for the orthogonal experiment at three levels. The SDS-PAGE was used to test the recombinant protein expression. The content of the fusion protein in the supernatant analyzed by standard SDS-PAGE figure method was determined as the effect indicator.The obtained data were analyzed statistically by SPSS 13. 0 software. The results showed that the recombinant vector p E-SUMO-PS was constructed successfully. And the fusion protein was expressed in the cytoplasm of E. coli in a soluble fraction. The quantity of fusion protein was the largest under the conditions of 30 ℃,0. 05 mmol / L concentration of IPTG induced for 6 hours. The intensity of impact from high to low was induction temperature,induction time and concentration of inducer. In this study,the best expression conditions were obtained through the orthogonal experiment,including 30 ℃ inducing temperature,six hours-inducing time and the0. 05 mmol / L concentration of IPTG. These provided an important reference valuable to subsequent purification and industrialization of the antimicrobial peptide plectasin.