非融合海参溶菌酶在大肠杆菌中的表达、纯化及酶活鉴定  

Expression,purification and activity identification of non-fusion recombinant Stichopus japonicus lysozyme in Escherichia coli

作　　者：张齐[1] 牛庆昌 姜琳 黄君 马俊[1] 丛丽娜[1] 李成[1] ZHANG Qi;NIU Qingchang;JIANG Lin;HUANG Jun;MA Jun;CONG Lina;LI Cheng(School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China)

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》2018年第2期84-88,共5页Journal of Dalian Polytechnic University

基　　金：国家海洋公益性行业科研专项(201405003-3);辽宁省自然科学基金重点项目(20170520043);辽宁省教育厅一般项目(L2014217)

摘　　要：利用实验室已构建好的表达载体pET32a(+)-SjLys,在基因工程菌RoSetta(DE3)plysS中表达重组融合海参溶菌酶rSjLys。利用融合的组氨酸标签,通过Ni 2+-NTA层析柱分离、纯化,获得高纯度的重组海参溶菌酶。利用重组牛肠激酶在22℃、pH 7.4、16h条件下对目的蛋白rSjLys的融合标签Trx-His-S tag进行完全酶切,通过Ni 2+-NTA层析柱纯化得到纯度大于99%的非融合的海参溶菌酶SjLys。实验结果表明,作为i型溶菌酶,与rSjLys比较,非融合海参溶菌酶对指示菌溶壁微球菌具有明显的抑菌作用。The recombinant fusion Stichopus japonicus lysozyme protein（rSjLys）was expressed in the RoSetta（DE3）plysS using the constructed expression vector pET32 a（＋）-SjLys.The recombinant Stichopus japonicus lysozyme protein rSjLys with fused histidine tag was isolated and purified by Ni 2＋-NTA chromatographic column.The fusion protein Trx-His-S tag of the target protein rSjLys was completely digested by recombinant bovine enterokinase at 22 ℃,pH 7.4 for 16 h.Then non-fusion Stichopus japonicus lysozyme protein（SjLys）was purified by Ni 2＋-NTA column,of which purity was 99%.The result showed that,as i-type lysozyme,SjLys has obvious bacteriostatic effect on Micrococcus lysodeikticus compared with rSjLys.

关 键 词：海参溶菌酶 重组蛋白 肠激酶酶切 抑菌活性 

分 类 号：TS254[轻工技术与工程—水产品加工及贮藏工程] Q786[轻工技术与工程—食品科学与工程]