厚壳贻贝抗菌肽myticusin-1的重组表达及功能分析

Recombinant Expression and Functional Analysis of the Antimicrobial Peptide Myticusin-1 of Mytilus coruscus

抗菌肽在无脊椎动物的先天免疫中起着重要作用。贻贝体内富含各种抗菌肽分子,其中,myticusin-1是厚壳贻贝体内发现的一种分子量较大的新型抗菌肽。天然myticusin-1在贻贝体内含量极低,因此采用大肠杆菌表达体系,结合密码子优化策略开展了厚壳贻贝myticusin-1的重组表达。表达产物经复性和纯化,所得重组myticusin-1表现出对革兰氏阳性菌、阴性菌以及真菌的广谱抑菌活性,其中,对革兰氏阳性菌的抑制活性较强。为进一步研究myticusin-1在厚壳贻贝体内的功能,采用实时荧光定量PCR技术,对厚壳贻贝分别在金黄葡萄球菌、溶藻弧菌和白色念珠菌的诱导下,其血细胞myticusin-1基因表达水平的时间曲线。结果表明厚壳贻贝myticusin-1基因表达对金黄葡萄球菌最敏感,且对金黄葡萄球菌的应激效应时间最持久。上述研究结果表明myticusin-1在厚壳贻贝体内可能参与对革兰氏阳性菌的免疫防御。

Antimicrobial peptides play important roles in innate immunity of invertebrates.Myticusin-1 is a novel antimicrobial peptide with a large molecular weight identified previously in Mytilus coruscus.Because of the low content of the natural myticusin-1 in mussels,the recombinant expression of myticusin-1 was performed using Escherichia coli expression system combined with codon optimization strategy.After renaturation and purification,the recombinant myticusin-1 showed broad spectrum antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and fungi.Among which,the recombinant myticusin-1 showed strong antimicrobial activities against Gram-positive bacteria.To further study the function of myticusin-1 in mussels,a real-time quantitative PCR was used to detect the expression in the hemocytes induced by Staphylococcus aureus,Vibrio alginolyticus and Candida albicans,respectively.The results showed that myticusin-1 gene expression showed most sensitivety to S.aureus and had the longest stress effect on it.These results suggest that myticusin-1 might be involved in defensing Gram-positive bacteria in M.coruscus.