重组海参溶菌酶N端可溶性表达与抑菌分析

The Souble Expression and Antibacterial Activity of N Terminus Region Lysozyme Gene from Sea Cucumber Stichopus japonicus

通过对海参(Stichopus japonicus)溶菌酶(SjLys)cDNA(Genbank accession no:EF036468)片段的分析,结果发现N端基因区域所对应的蛋白质序列中含有糖苷酶活性。因此利用RT-PCR技术以其为模板,扩增出长度为174 bp的溶菌酶N端(SjLys-N)基因,将其亚克隆至原核表达载体pET-32a(+),构建重组质粒pET-32a(+)-SjLys-N,再转化至大肠杆菌Rosetta(DE3)pLysS,成功地构建了重组SjLys-N的基因工程菌。该工程菌在改造的LB培养基和最适的发酵条件下经IPTG诱导后,表达约23 kD的重组SjLys-N蛋白。此外,它还能以可溶性的形式表达,占总菌体蛋白约46%。经过Western blotting分析,重组SjLys-N在23 kD左右能够与penta-his抗体发生特异性免疫反应,结果表明重组SjLys-N得到正确的表达。最后对纯化后的重组SjLys-N进行了抑菌特性的分析,结果发现它对溶壁微球菌和副溶血弧菌有较高的抑菌活性。上述结果将为进一步研究海参溶菌酶的基因结构与功能之间的关系提高参考。

We analysed the gene of Stichopus japonicus lysozyme （SjLys） cDNA （GenBank accession no： EF036468） by bioinformatics. The result showed that the amino acid region of N terminus of SjLys contains glycosidase activity. Thereforce, the gene fragment of N terminus of SjLys （SjLys-N） was amplified by RT-PCR method from its cDNA. As a result, the target gene was obtained at the length of 174 bp. Next, the DNA fragment of SjLys-N was subclone into the expression vector of pET-32a （＋） to construct the recombinant plasmid of pET32a （＋）-SjLys-N. Then the recombinant plasmids were transformed into Escherichia coli Rosetta （DE3）pLysS to gain the genetically engineering strain pET-32a （＋）-SjLys-N/E. coli Rosetta （DE3）pLysS. After IPTG induction with the modified LB media and optimal fermentation conditions, the genetically engineering strain could highly express the recombinant protein SjLys-N of 23 kD. Moreover, the recombinant SjLys-N could express in solube form, which was taken up 46 % of the total protein. After Western blotting analysis, it was found that the recombinant SjLys-N had a specific immune response with Penta-His antibody at the position of about 23 kD. Therefore, it is evidence that the recombinant SjLys-N must be the target protein. The antibacterial activity of the purified recombinant SjLys-N was analyzed. The SjLys-N protein displayed inhibitive effect on the growth of the Micrococcus lysodeikticus and Vibrio parahaemolyticus.In conclusion, these results will provide a foundation to further study the relationship between gene structure and function of the sea cucumber lysozyme.