树蛙抗菌肽Cathelicidin在毕赤酵母中的表达及抑菌活性分析  

Expression of Tree Frog(Rhacophorus) Cathelicidin Peptide in Pichia pastoris and Its Antibacterial Activity Analysis

作　　者：王莲哲 刘士俊 刘佳乐 洪军 WANG Lian-Zhe;LIU Shi-Jun;LIU Jia-Le;HONG Jun(College of Life Science and Engineering,Henan University of Urban Construction,Pingdingshan 467000,China)

机构地区：[1]河南城建学院生命科学与工程学院,平顶山467000

出　　处：《农业生物技术学报》2021年第1期67-72,共6页Journal of Agricultural Biotechnology

基　　金：河南省高等学校重点科研项目计划(19A180011)。

摘　　要：树蛙(Rhacophorus)抗菌肽Cathelicidin是一类具有广谱抗微生物活性的小分子多肽,在医疗、畜牧等方面有潜在应用价值,但其真核表达少有报道。本研究根据毕赤酵母(Pichia pastoris)的密码子偏爱性进行优化,合成树蛙抗菌肽Cathelicidin的目的基因,与表达载体pPIC9K连接后电击转化至毕赤酵母GS115,通过高浓度G418筛选高拷贝酵母转化子,通过PCR和反转录PCR (reverse transcription PCR, RTPCR)验证,获得的阳性转化子使用1%甲醇诱导表达72 h后,收集培养液上清液进行体外抑菌活性检测及毒性分析。结果表明,树蛙抗菌肽Cathelicidin在毕赤酵母GS115中获得成功表达,分泌表达的抗菌肽对革兰氏阳性菌金黄色葡萄球菌(Staphylococcus aureus)和革兰氏阴性菌大肠杆菌(Escherchia coli)具有抑菌活性,最小抑菌浓度(minimal inhibitory concentration, MIC)分别为(1.175±0.002)和(2.35±0.001)μg/mL,最小抑菌浓度下没有细胞溶血性。本研究为新型树蛙抗菌肽的工业生产提供基础数据支持。Tree frog(Rhacophorus) antimicrobial peptide Cathelicidin is a kind of small molecular peptide with antibacterial activity, which has potential application value in medical treatment, animal husbandry and some other aspects, but its eukaryotic expression is rarely reported. In this study, tree frog antimicrobial peptide Cathelicidin gene was synthesized based on the preferential codon usage of Pichia pastoris, then ligated to the expression vector pPIC9 K and transformed into P. pastoris GS115 by electroporation. The positive transformants containing multi-copy gene insertions were screened using high concentration G418 and confirmed by PCR and reverse transcription PCR(RT-PCR). The recombinant Cathelicidin was induced for 72 h with 1.0%methanol, and the supernatant of culture medium was collected for in vitro antibacterial activity detection and toxicity analysis. The results showed that Cathelicidin was expressed successfully in P. pastoris with antibacterial activity against gram-positive bacteria(Staphylococcus aureus) and gram-negative bacteria(Escherchia coli), the minimal inhibitory concentration were(1.175±0.002) and(2.35±0.001) μg/mL respectively, and it was no hemolysis under the minimum inhibitory concentration. This study provides basic data support for industrial production of a new tree frog antimicrobial peptide.

关 键 词：抗菌肽Cathelicidin 毕赤酵母 抑菌活性 分泌表达 

分 类 号：S874[农业科学—畜牧兽医]