东北林蛙皮肤抗菌肽基因在毕赤酵母中表达条件的优化

Optimization of Pichia pastoris Expression of Rana dybowskii Antimicrobial Peptide Gene

目的:确定毕赤酵母表达东北林蛙皮肤抗菌肽dybowskin-1ST基因的最佳诱导表达条件。方法:本研究将PCR合成的dybowskin-1ST基因克隆到毕赤酵母表达载体pPIC9K中,构建pPIC9K-dybowskin-1ST诱导型表达载体。表达载体经线性化后转化巴斯德毕赤酵母GS115,应用BMMY培养基诱导表达。采用单一变量法及正交试验法,对甲醇诱导的浓度、时间和温度进行了优化,通过SDS-PAGE电泳分析和体外抑菌试验检测目的肽表达丰度。结果:最佳诱导条件:甲醇终浓度为0.5%、诱导时间为72 h、诱导温度为28℃,在此条件下表达产物SDS-PAGE电泳条带灰度值为117,抑菌环直径为28.16mm,可为规模化的抗菌肽表达提供参考数据。

Our study objective was to determine the optimal induction condition for Pichia pastoris expression of dybowskin- 1ST gene of Rana dybowskii skin antimicrobial peptides.The dybowskin - 1ST gene was synthesized by PCR and cloned into Picha pastoris expression vector pPIC9K to generate inducible expression vector pPIC9K - dybowskin - 1ST.The linearized plasmid pPIC9K - dybowskin - 1ST was transformed into Pichia pastoris yeast GS115.Expression was induced by BMMY culture medium.Using the single variable method and orthogonal experiment method,we optimized inducing conditions by changing the temperature,the final concentration of methanol and the induction time.The optimal conditions were determined in vitro by SDS - PAGE electrophoresis detection and antibacterial tests.For the optimal induction conditions,methanol final concentration,induction time,and induction temperature were 0.5%,72 h and 28℃,respectively.Under these conditions, the gray value of SDS - PAGE bands of expression product was 117,and the diameter of the inhibition ring was 28.16 mm.These results provide reference data for large - scale expression of antimicrobial peptides.