Recombinant Expression Of Sea Cucumber Lysozyme Its C-terminal Region From Stichopus Japonicas In And Analysis Of Antimicrobial Activity

Lysozyme is a kind of alkaline proteinase. It specially functions as hydrolysing the β-1,4glycosidic bond between N-acetylmuramic acid and N-accetylgkucosamine of the peptidoglycan which is an important constituent of gram-positive cell wall and leading the cell death with consequent lysis of the bacterial cell wall. Lysozyme is widely distbibuted in animal plant and microorganism tissues and plays an important role in the immune system of living organism. According to analyse the lysozyme of sea cucumber, it is found that it contains glycosidase activity and isopeptidase activity. However, there is no report yet about the genetic engineering method to recombinate the C terminal region Stichopus japonicas lysozyme in Pichia pastoris expression system. This is very unfavorable to the development of sea cucumber i type lysozyme of sea cucumber. The project mainly focused on recombinant expression of lysozymeof sea cucumber (SjLys) and the C-terminal region of the sea cucumber lysozyme from Stichopus japonicas (SjLys-C) in the yeast Pichia pastoris.The DNA fragment of SjLys-C was subcloned into the eukaryotic expression vector of pPIC9K to construct the recombinant plasmid of pPIC9K-SjLys-C. Two recombinant plasmid, pPIC9K-SjLys (previously constructed in our lab) and pPIC9K-SjLys-C were linearized by BglⅡ and transformed into P. pastoris GS115by electroporation, respectively. The recombinant genetic engineering strains, P. pastoris GS115/pPIC9K-SjLys and P. pastoris GS115/pPIC9K-SjLys-C, were screened by the YPD plate which containing antibiotics G418, respectively. The phenotype His+Muts of transformants were screened out by MD and MM plates. As a result, six recombinant transformants with multiple copies of SjLys gene and four with multiple copies of SjLys-C gene were obtained after inducing by1%methanol, all of these transformants possessing antibacterial activity.The results showed that the highest expression level with a wide spectrum of antibacterial activity was induced by methanol at72h. The recombinant protein SjLys-C and Sjlys displayed inhibitive effect on the growth of the Gram positive and negative tested strains, especially against Vibrio parahaemolyticus and Pseudomonas aeruginosa which usually caused serious diseases in aquatic animal health. The SjLys and SjLys-C genes were successfully expressed in Pichia pastoris. This will porvide a basis to make a further research in the structure and function of SjLys and SjLys-C and the expression in Pichia pastoris. It also provides a basis for further large-scale industrial production.