人溶菌酶基因在毕赤酵母中的表达及其抗菌活性检测  

Expression of the human lysozyme in Pichia pastoris and antibacterial activities test

作　　者：夏清风[1] 侯英敏[1] 曹芳[1] 金朝霞[1] 

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2011年第5期346-349,共4页Journal of Dalian Polytechnic University

基　　金：大连市优秀青年人才基金项目(2009J22DW036)

摘　　要：将PCR扩增的人溶菌酶编码基因克隆到毕赤酵母分泌型表达载体pPIC9K中,双酶切和测序鉴定表明,重组载体构建正确。电转化毕赤酵母GS115菌株,通过G418筛选获得高拷贝重组菌株后,用甲醇诱导表达;通过SDS-PAGE检测证明上清中有目的蛋白,表达产物与预期大小的人溶菌酶蛋白分子质量15ku一致,其表达量占分泌总蛋白的32%。体外抑菌实验证实重组人溶菌酶蛋白对溶壁微球菌、金黄色葡萄球菌、大肠杆菌和枯草杆菌均有明显的抑菌作用,表明在毕赤酵母中成功表达了重组人溶菌酶。The HLY coding gene was cloned into P. pastoris expression vector pPIC9K. The pPIC9K/HLY was verified by two enzyme digestion and sequencing and then transferred into P. pastoris GSl15 strain by electroporation. The recombinant strains were isolated by G418. The results of SDS-PAGE demonstrated that the fermentation supernatant contained the recombinant protein up to 32% of total proteins and its molecular weight is 15 ku. In vitro the recombinant protein has certain antimicrobial activity against M. lysodei/eticus, E. coli, S. aureas and B. subtilis, which show that the successful expression of the recombinant HLY in P. pastoris and thus provide the basis for its large-scale production using P. pastoris system.

关 键 词：人溶菌酶 毕赤酵母 PPIC9K 表达 

分 类 号：TQ127.2[化学工程—无机化工]