重组海参溶菌酶C端基因(SjLys-C)的可溶性表达和抑菌活性分析

为进一步研究海参(Stichopus japonicus)溶菌酶基因(Sjys)(Genbank登录号:EF036468)中不司片段表达产物的生物特性,本研究通过对其cDNA片段的分析,发现C端基因区域所对应的蛋白质序列中含有非酶活性.根据已知的海参溶菌酶的cDNA序列,设计山含有Nco Ⅰ和EcoR Ⅰ酶切位点的特异性引物,从新鲜的海参肠中提取总RNA,以其为模板利用RT-PCR扩增出长度为259 bp的溶菌酶C端(SjLys-C)基因.将该目的基因连接到pET-32a(+)载体上,构建重组质粒pET-32a(+)-SjLys-C,再转化至大肠杆菌(Escherichia coli)Rosetta(DE3)pLysS,成功地构建了重组蛋白SjLys-C的基因工程菌.利用该工程菌诱导发酵,结果显示它能高效表达出26 kD左右的重组蚩白SjLys-C.经过Western blot分析,该重组蛋白在26 kD左右能够与Penta-His抗体发生特异性免疫反应.对纯化的重组蛋白SjLys-C进行了抑菌特性的分析,结果发现它对溶壁微球菌(Micrococcus lysodeikticus)和副溶血弧菌(Vibrio parahae molytic us)有较高的抑菌活性.此外,将该重组蛋白经100℃、40 min处理后,其抑菌能力提高了5％～21％.研究结果表明,重组蛋白SjLys-C基因工程菌能够制备出具有可溶性的、并具有抑菌活性的重组蛋白SjLys-C,在农业和医药等行业中有潜在应用和开发价值.

The Soluble Expression and Antibacterial Activity of C Terminus Region Lysozyme Gene(SjLys-C) from Sea Cucumber(Stichopus Japonicus)

In order to characterize the antibacterial activity of gene products expressed by several gene fragments from the gene of Stichopus japonicus lysozyme (SjLys), its cDNA (GenBank accession No. EF036468) was analysed by bioinformatics. The result showed that the amino acid region of C terminus of SjLys contained non-enzymatic activity. Therefore, a pair of primers including NcoⅠ and EcoRⅠrestriction sites were designed according to the cDNA sequence of SjLys and the gene fragment of C terminus of SjLys (SjLys-C) was amplified by RT-PCR from the total RNA of the S. japonicus intestine. As a result, the target gene was obtained at a length of 259 bp fragment. The fragment of SjLys-C was subcloned into the expression vector of pET-32a(+) to construct the recombinant plasmid of pET-32a(+)-SjLys-C. Then the recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS to gain the genetically engineering strain pET-32a(+)-SjLys-C/ E. coli Rosetta(DE3)pLysS. We used the strain to induce and express the recombinant protein SjLys-C. The result showed that the genetically engineering strain could highly express the recombinant protein of 26 kD. Moreover, the recombinant protein SjLys-C could express in solube form, which was taken up 70% of the total protein. Western blotting analysis found that the recombinant protein SjLys-C had a specific immune response with Penta-His antibody at the position of about 26 kD. Therefore, it is evidence that the recombinant protein SjLys-C must be the target protein. The antibacterial activity of the purified recombinant protein SjLys-C was also analyzed. The recombinant protein SjLys-C displayed inhibitive effect on the growth of the Micrococcus lysodeikticus and Vibrio parahaemolyticus. In particular, the heat-treated recombinant protein SjLys-C inhibitive activities were enhanced from5% to 21% at 100℃and 40 min treatment. These results showed that the strain pET-32a(+)-SjLys-C/E. coli Rosetta(DE3)pLysS could produce recombinant protein SjLys-C in a soluble form and exhibit the potent antibacterial activity. Therefore, the product will be widely used in agriculture and medicine and has great market potential and development value.