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

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.