[Tandem expression and activity determination of antibacterial peptide Spinosan-C from Paa spinosa]

Antibacterial peptide can be easily degraded by protease and has the lethal effect on the host Escherichia coli. In order to solve these problems and further improve the expression ability of the Escherichia coli system, the antimicrobial peptide Spinosan-C of Paa spinosa was studied. First, the codon of Spinosan-C was optimized according to E. coli codon usage frequency. Then, the 8 multimeric Spinosan-C gene (8×Spinosan-C) was synthesized and cloned into prokaryotic expression vector pET-28a. The fusion antimicrobial peptide 8×Spinosan-C was further highly expressed in Escherichia coli strain Rosetta. The recombinant 8×Spinosan-C protein was then purified and cleaved specially by formic acid to generate the Spinosan-C monomer. Antibacterial test in vitro suggested that the cleaved Spinosan-C monomer had antibacterial bioactivity against the test bacteria. This study provides a technical reference for the largescale preparation of frog antimicrobial peptides.为克服抗菌肽易被蛋白酶降解及对宿主大肠杆菌的杀伤作用，并进一步提高大肠杆菌系统的表达能力，以棘胸蛙Paa spinosa 抗菌肽Spinosan-C 为研究对象，按照大肠杆菌密码子利用频率进行密码子优化，设计合成8 拷贝的串联8×Spinosan-C 基因，将合成的串联基因克隆到大肠杆菌表达载体pET-28a，利用大肠杆菌感受态细胞Rosetta 进行原核表达，获得高效表达的串联8×Spinosan-C 重组蛋白，用甲酸专一性切割得到抗菌肽Spinosan-C单体。体外抑菌试验表明，切割后的抗菌肽Spinosan-C 单体对测试菌生长具有抑制作用，为蛙类抗菌肽的规模化制备提供了参考。.