Integrative Expression of Stichopus japonicus Lysozyme Gene in Bacillus subtilis

Abstract

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

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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