海刺参i型溶菌酶基因的重组表达及抑菌谱分析

Recombinant expression and antibacterial activity of i-type lysozyme from sea cucumber Stichopus japonicus

将本实验室已分离到的海刺参（Stichopus japonicus）i型溶菌酶的基因（GenBank Accession No.EF036468）亚克隆进原核表达载体pET32a（＋）中,构建重组质粒pET32a（＋）-SjLys,转化至大肠杆菌BL21（DE3）pLysS。阳性克隆子经诱导表达、亲和纯化和透析复性,得到了酶活力为19.2 U/mg的重组SjLys（rSjLys）,并对rSjLys进行了抑菌谱测定。结果表明,rSjLys对革兰氏阳性菌和阴性菌均有抑制作用,尤其对常见的海洋致病菌副溶血弧菌和铜绿假单胞菌具有很强的抑菌活性。更为显著的结果是,rSjLys经100oC、40 min加热处理后,失活的rSjLys对革兰氏阳性菌和阴性菌的抑菌能力高于rSjLys的9%~25%。上述结果表明,海刺参溶菌酶是一种具有糖苷酶活性和非酶抑菌活性的特殊的i型溶菌酶,且具有很广的抑菌活性,在海刺参机体先天免疫系统中是一个重要的效应分子。

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 pET-32a （＋） 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 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 was 19.2 U/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, rSjLys had a strong inhibitive activity on Vibrio parahaemolyticus and Pseudomonas aeruginosa, both the most common pathogenic bacteria in the marine animals. 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 wide 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.