海参溶菌酶基因在枯草芽孢杆菌WB600中的整合及表达

摘要/Abstract

摘要： 采用枯草芽孢杆菌WB600(Bacillus subtilis WB600)在体外表达海刺参溶菌酶(Stichopus japonicus lysozyme，SjLys)蛋白。通过构建克隆质粒pMD18-P43-SjLys，将B. subtilis自身强启动子P43序列和已分离得到的SjLys基因(GenBank登录号EF036468)连接(P43-SjLys)。经BamH I/EcoR I酶切，将P43-SjLys片段连接到B. subtilis整合载体pDG1730上，构建整合重组质粒pDG-P43-SjLys。经Xho I酶切处理，线性化的pDG-P43-SjLys质粒转化B. subtilis WB600细胞。P43-SjLys片段通过同源双交换重组整合到B. subtilis WB600染色体上，成功得到具有稳定遗传的基因工程菌B. subtilis WB600/P43-SjLys。经SDS-PAGE和抑菌试验分析表明，培养60 h后，B. subtilis WB600/P43-SjLys能够表达可溶的，对常见的海洋细菌溶壁微球菌和金黄色葡萄球菌具有较强的抑菌活性的SjLys蛋白。首次在B. subtilis表达系统中得到可溶且具有酶活功能的SjLys蛋白，为SjLys的生产提出了一种具有可行性的和潜力的新方法。

关键词: 海参溶菌酶, 枯草芽孢杆菌, 启动子P43, 同源重组, 整合表达

Abstract: In this study, we cloned promoter P43 of B. subtilis in the front of Lysozyme from Stichopus japonicus(SjLys) gene by constructing the recombinant plasmid pMD18-P43-SjLys. Then we obtained the integration plasmid pDG-P43-SjLys of B. subtilis by subcloning the P43-SjLys fragment into the plasmid of pDG1730. We transformed the linear integration plasmid digested by Xho I into B. subtilis WB600, and selected the re-combinant strain by Spc resistance screening and amylase activity negative screening. After incubation at 37℃, 220 r/min in LB medium, the cellular lysate of this strain was detected by the SDS-PAGE assay. The results demonstrated that B. subtilis WB600/P43-SjLys successfully expressed soluble SjLys after incubated for 60 h. The cellular lysate of B. subtilis WB600/P43-SjLys displayed inhibitive effect on the growth of the Micrococcus lysodeikticus and Staphylococcus aureus. In this study, we expressed SjLys gene integratedly in B. subtilis for the first time, and proposed a potentially new way of producing SjLys protein.

Key words: Lysozyme of Stichopus japonicus, Bacillus subtilis, Promoter 43, Homologous recombination, Integrative expression

李丹, 李成, 孙璐, 邹丹, 刘志文, 丛丽娜. 海参溶菌酶基因在枯草芽孢杆菌WB600中的整合及表达[J]. 生物技术通报, 2014, 0(4): 139-146.

Li Dan, Li Cheng, Sun Lu, Zou Dan, Liu Zhiwen, Cong Lina. Integrative Expression of Stichopus japonicus Lysozyme Gene in Bacillus subtilis[J]. Biotechnology Bulletin, 2014, 0(4): 139-146.

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