东北林蛙皮肤抗菌肽dybowskin-1ST的表达、纯化及抑菌活性分析

  【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。

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

【Objective】 The aim of this study is to express Rana dybowskii antimicrobial peptide dybowskin-1ST 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-1ST 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-1ST 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.