颗粒裂解肽G13结构域的重组表达及蛋白质结构预测

Recombinant expression and protein structure prediction of G13 domain from granulysin

基因工程构建表达是获得抗菌肽的一种成本较低的方法,本实验人工合成G13结构域编码DNA序列,PCR扩增后,用T-A克隆法与pBAD/TOPO ThioFusion表达载体连接,通过PCR鉴定筛选出正确重组质粒,在大肠杆菌Top10中对目的蛋白进行表达,大肠杆菌工程菌经阿拉伯糖诱导后取样,用SDS-PAGE检测表达情况,采用生物信息学方法对表达蛋白的结构特征进行模拟分析。结果显示:目的蛋白在原核系统中实现了高效表达,表达量高达67%以上,主要以包涵体形式表达。蛋白结构预测结果显示,目的蛋白原有的α螺旋活性结构无改变,从而为抗菌肽高效生产提供了有效可靠的研究途径。

Genetic engineering construction expression is a lower cost approach to get new antimicrobial peptides （AmPs）. The G13 domain coding sequence was synthesized and amplified by PCR. Using T-A cloning technique, the PCR product was cloned into the pBAD/TOPO ThioFusion vector. The correct recombinant plasmid was screened out with PCR. After inducing with L-arabinose, the total protein of the engineered E. coli was extracted and detected by SDS-PAGE. The fusion protein property like second structure was estimated by PHD and PROF software. High expression of the fusion protein was obtained. The predicted structure showed that second structure of the G13 domain still preserved α helix.