Over-expression Of The Lysozyme Gene In E.Coil From The Sea Cucumber Stichopus Japonicus

Sea cucumbers are a typical representative of marine invertebrates. In recent years, the study of sea cucumber mainly concentrated on the breeding, deep processing of products and medicinal activity, whereas the molecular investigation of sea cucumber is less reported. In this project, the over-expression of the lysozyme gene from the intestine of the sea cucumber was carried out in Escherichia coli and the recombinant lysozyme was examined on its antimicrobial activities. The aim of the study is to provide a theoretical foundation on sea cucumber growth, metabolism and autolysis phenomenon.Lysozyme is widely distributed among eukaryotes and prokaryotes. It catalyzes the hydrolysis of bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogen. The commercial lysozyme, mainly obtained from hen egg white, only functions to gram-positive bacteria, so its application was limited. In recent years, researches of lysozymes from many sources have been done widely, but the report of lysozymes in marine organisms was very little.All of the lysozyme cDNAs of Stichopus japonicus contains an open reading frame (ORF) of438bp. The438bp ORF encodes145amino acid residues, including125amino acid residues and21amino acid residue of signal peptide.The cDNA of an i type lysozyme was cloned from Stichopus japonicus (named as SjLys). The DNA fragment of the mature SjLys was subcloned into expression vector of pET32a (+) to construct the recombinant plasmid of pET32a (+)-SjLys. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) pLysS and induced by isopropylthio-β-D-galactoside (IPTG). The best conditions for inducible expression are that No.2strain is induced by0.5mM IPTG for4h in30℃. The recombinant protein expressed as inclusion bodies was denatured, partially purified and refolded to be an active form. The bacteriolytic activity of recombinant protein purified by the metal-chelating was19.2U/mg.The antibacterial activity of the purified recombinant SjLys (rSjLys) was analyzed. The rSjLys protein displayed inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. In particular, the rSjLys had a strongly inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa which were the most common pathogenic bacteria in the marine animals. A remarkable finding was the heat-treated rSjLys exhibited more potent activities against all tested bacteria. These results indicated that the S. japonicus lysozyme was the enzyme with combined enzymatic (glycosidase) and non-enzymatic antibacterial action, and it had a widely antibacterial spectrum. Therefore, it is suggested that the S. japonicus lysozyme should be one of the important molecules against pathogens in the innate immunity of sea cucumbers.