东北林蛙皮肤抗菌肽dybowskin-1ST的表达、纯化及抑菌活性分析

Expression,Purification and Antibacterial Activity Analysis of Rana dybowskii Antimicrobial Peptide Dybowskin-1ST

【目的】利用毕赤酵母菌真核表达系统重组表达东北林蛙皮肤抗菌肽dybowskin-1ST,分析评价dybowskin-1ST的抑菌活性。【方法】采用TRIzol法提取蛙皮总RNA,经RT-PCR扩增获得抗菌肽dybowskin-1ST基因,将目的基因克隆到毕赤酵母菌分泌型表达质粒pPIC9K中,得到重组质粒pPIC9K-1ST。经电转化将pPIC9K-1ST转入到毕赤酵母菌GS115中,获得高效表达dybowskin-1ST的重组菌株。以甲醇诱导重组菌株,上清液经SDS-PAGE检测表明有目的蛋白dybowskin-1ST分泌表达。目的蛋白经Ni-agarose色谱柱分离纯化后,对大肠埃希氏菌、肺炎克雷伯氏杆菌、金黄色葡萄球菌、绿脓杆菌、肠炎沙门氏菌和蜡状芽孢杆菌进行抑菌活性分析。【结果】成功构建了东北林蛙皮肤抗菌肽dybowskin-1ST的毕赤酵母真核表达系统,且dybowskin-1ST对革兰氏阳性菌和革兰氏阴性菌均有良好的抑菌效果。【结论】东北林蛙皮肤抗菌肽dybowskin-1ST具有良好的广谱抗菌活性和潜在的研发价值。

[ Objective ] The aim of this study is to express Rana dybowskii antimicrobial peptide dybowskin-1 ST via Pichia pastoris system and to analyze its antibiotic activity. [ Method ] The total RNA of Rana dybowskii skin was extracted by TRIzol method. The antimicrobial peptide dybowskin-1 ST DNA were obtained by RT-PCR and cloned into expression vector pPIC9K, and recombinant pPIC9K-1ST was obtained. Then, the pPIC9K-1ST was electrotransformed into Pichia pastoris GS115, and the recombinant strain expressing dybowskin-1ST was generated. The recombinant strain was induced by the methanol and the interest protein was expressed via SDS-PAGE analysis. The interest protein was purified with Ni-agarose and its antimicrobial activity was detected using Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, Pseudomonas aeruginosa and Klebsiella pneumoniae. [ Result ] The recombinant Pichia Pastoris system expressing Rana dybowskii antimicrobial peptide dybowskin-1 ST was well-constructed and the peptide dybowskin-1ST showed significant antimicrobial activity on gram-positive bacteria and gram-negative bacteria. [Conclusion] Rana dybowskii antimicrobial peptide dybowskin-1ST has a broad range of antimicrobial activity and a number of potential applications.