金黄色葡萄球菌噬菌体qdsa001内溶素的特性预测及克隆表达

为获得噬菌体qdsa001的内溶素，本实验首先利用多种在线软件对内溶素lys56（GenBank：ARQ95812.1）的理化性质、信号肽、跨膜域和结构域等特性进行分析，然后选择克隆表达法制备目的蛋白。将合成的目的基因插入原核表达载体pET-30a中，构建重组质粒pET-30a-lys56，转化至大肠杆菌Rosetta（DE3）中，获得表达稳定的基因工程菌，经异丙基硫代-β-D-半乳糖苷（isopropyl-β-D-thiogalactopyranoside，IPTG）诱导表达，利用镍琼脂糖亲和层析纯化表达产物。经在线软件预测结果显示，该蛋白分子质量为35.1 kDa，无信号肽和跨膜域，仅含有一个Ami_2结构域。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示重组蛋白表达成功，且以可溶性蛋白的形式存在于细胞破碎上清液，分子质量约35?kDa，与预期蛋白大小一致。重组蛋白最佳表达条件为：IPTG浓度0.5?mmol/L，20?℃过夜诱导。

Characteristics and Expression of Endolysin from Staphylococcus aureus Bacteriophage qdsa001

This study aimed to obtain the endolysin lys56 encoded by Staphylococcus aureus phage qdsa001. First of all, the characteristics (physicochemical properties, signal peptide, transmembrane helices and structure domains) of lys56 were predicted by several online software. Subsequently, the target protein was obtained by cloning and expression. For this purpose, the target gene was synthesized and inserted into a prokaryotic expression vector pET-30a to construct recombinant plasmid pET-30a-lys56, which was subsequently transformed into E. coli Rosetta (DE3). Ni-IDA affinity chromatography was applied to purify the recombinant protein. The results showed that lys56 had a relative molecular mass of 35.1 kDa without signal peptide or transmembrane domain and contained only one Ami_2 domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant protein was successfully expressed in the cell supernatant with a molecular mass of about 35 kDa, which was consistent with the expected size. Optimal expression of the endolysin lys56 was induced by 0.5 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 20 ℃.