日本对虾c型溶菌酶的高效重组表达及产物分析

从日本对虾(Marsupenaeus japonicus)血液中提取总RNA,根据GenBank已登录的该cDNA序列(AB080238),通过RT-PCR技术扩增出日本对虾溶菌酶(MjLys)成熟肽基因。该基因完整的开放阅读框为477 bp,编码158个氨基酸(aa),前18 aa为信号肽,成熟肽由140 aa组成,分子量为16.4 kD,理论等电点(pI)为8.80。经分析表明,该基因含有一个完整的c型溶菌酶结构域(1-130 aa),包括c型溶菌酶特有的两个活性中心Glu33和Asp50,以及8个保守结构Cys残基。将MjLys成熟肽基因亚克隆至原核表达载体pET-32a(+),在大肠杆菌细胞BL21(DE3)pLysS中诱导发酵,实现了重组MjLys蛋白的高效表达,并测定了该重组蛋白对几种细菌的抑菌活性。结果表明,重组日本对虾溶菌酶对革兰氏阳性菌金黄色葡萄球菌和溶壁微球菌均有显著的溶菌活性。

High Level Recombinant Expression of c-type Lysozyme from Marsupenaeus japonicus and Product Analysis

The total RNA from blood of Marsupenaeus japonicus was extracted based on the logined cDNA sequence in GenBank(AB080238).The complete lysozyme gene from M.japonicus by RT-PCR was cloned and named Mj-lysozyme(MjLys in short).The open reading frame of the gene consists of 477 bp and encoded 158 amino acids(aa),including a signal peptide of 18 aa and a mature peptide of 140 aa.The molecular weight of the mature protein was of 16.4 kD,and the isoelectric point is 8.80.It was found that structure of the MjLys gene contains a domain(residue 1-130 aa) of conserved c-type lysozyme,including the two catalytic residues Glu33 and Asp50 and the eight cysteine residue motif.The gene of mature peptides of MjLys was subcloned into the expression vector pET-32a(+) and transformed into E.coli BL21(DE3) pLysS.As a result,the recombinant protein of MjLys was strongly expressed.The present study suggested the MjLys possesses significant activities against Gram-positive bacteria Micrococcus lysodeikticus and Staphyloccocus aureus.