Integrative Expression Of Stichopus Japonicus Lysozyme Gene In Bacillus Subtilis And Analysis Of The Antibacterial Activity Of The Recombinant Protein

Lysozyme from Stichopus japonicus(SjLys)plays an important role in the immune system,and displays potent inhibitive effect on the growth of pathogenic bacteria.Bacillus subtilis as a new system of engineering bacterium has many advantages,including endotoxin-free,strong protein secretion ability.So it may replace E.coli expression system which is widely used before to build a sea cucumber lysozyme engineering bacterium,to avoid the presence of defects such as pathogenic,containing endotoxin(LPS)and the formation of inclusion bodies in E.coli system.In this study,we cloned promoter P43 of B.subtilis in the front of 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 XhoI into B.subtilis WB600,and selected the re-combinant strain by Spc resistance screening and amylase activity negative screening.After incubating at 37?,220 r/min in LB medium,the cellular lysate of the 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.