肠出血性大肠埃希菌噬菌体裂解酶的毕赤酵母重组表达及裂解活性分析  

Pichia pastoris recombinant expression and lysis activity of lysin of enterohemorrhagic Escherichia coli bacteriophage

作　　者：孙利厂[1] 庞茂达[1] 何灵尘 周艳[1] 何涛[1] 王冉[1] SUN Lichang;PANG Maoda;HE Lingchen;ZHOU Yan;HE Tao;WANG Ran(Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology/Key Laboratory of Control Technology and Standard for Agro-Product Safety and Quality, Ministry of A gricultrue and Rural Affairs/Key Laboratory of Animal-Derived Food Safety of J iangsu Province/ Institute of Food Safety and Nutrition,J iangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院农产品质量安全与营养研究所/省部共建国家重点实验室培育基地--江苏省食品质量安全重点实验室/农业农村部农产品质量安全控制技术与标准重点实验室/江苏省畜禽产品安全性研究重点实验室,南京210014

出　　处：《扬州大学学报（农业与生命科学版）》2018年第4期1-6,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：江苏省基础研究计划(自然科学基金)项目(BK20160585);江苏省农业科技自主创新基金项目[CX(15)1008];农业农村部畜禽产品病原菌和寄生虫污染风险评估项目(GJFP201800703)

摘　　要：为研究噬菌体V-EcoM-C1所编码裂解酶LysC1的裂解活性,在酵母菌GS115中重组表达裂解酶LysC1,并检测裂解酶LysC1对大肠埃希菌的裂解活性。结果表明:通过甲醇诱导后,含有裂解酶基因LysC1的重组表达质粒pPIC9K-LysC1能够在酵母菌GS115中表达,重组蛋白LysC1的分子量约为25ku,以外分泌方式表达,经HIS-trap HP(GE)亲和层析柱纯化后可获得有活性的重组蛋白LysC1,且该裂解酶对多种不同O抗原类型的肠出血性大肠埃希菌均具有较高的裂解活性,其最小抑菌浓度(MIC)为100μg·mL^(-1)。这一研究利用酵母表达系统成功表达出大肠埃希菌噬菌体裂解酶,且该裂解酶无需穿孔素等破膜因子的辅助,可直接裂解大肠埃希菌,为肠出血性大肠埃希菌的防控提供了新的思路和途径。This work aims to express the lysin gene LysC1 in Pichia pastoris and determine the antibacterial activity of the expressed product.The cloned LysC1 gene fragment was ligated to pPIC9 Kvector to construct a recombinant expression vector pPIC9 K-LysC1,which was transformed into competent P.pastoris GS115.Recombinant fusion protein LysC1 was induced with methanol,the expressed product was identified by SDS-PAGE,and antibacterial activity was determined by minimal inhibitory concentration.The results indicated that LysC1 was successfully expressed in P.pastoris,and LysC1 demonstrated antibacterial activity against different serotypes EHEC(MIC:100μg·mL^-1).LysC1 is a novel broad spectrum antibacterial lysin,which has the capacity to be a new generation antimicrobial agent.

关 键 词：肠出血性大肠埃希菌 噬菌体 裂解酶 

分 类 号：S852.612[农业科学—基础兽医学]