李斯特菌噬菌体裂解酶CBD蛋白的克隆与表达

Cloning and expression of Listeria phage endolysin CBD protein

单核细胞增生李斯特菌噬菌体的裂解酶中细胞壁结合结构域(CBD)能够特异识别李斯特菌细胞。本研究将CBDP40基因扩增后插入p ET32a(+)载体,导入大肠杆菌表达系统,获得重组菌株,并对其进行IPTG诱导表达,分析重组蛋白的活性。聚丙烯酰胺凝胶电泳(SDS-PAGE)表明,经0.1 mmol/L IPTG诱导的重组大肠杆菌BL21(DE3)在38 ku处有明显诱导条带,与预期蛋白分子量相符。经过超声细胞破碎,重组蛋白CBDP40分布在上清液中,通过镍离子螯合亲和层析纯化重组蛋白,洗脱后的蛋白浓度为0.908 mg/m L。经菌体结合和间接ELISA验证,重组蛋白CBDP40具有生物学活性,即对李斯特菌具有识别功能。CBD蛋白的重组表达、纯化及其活性验证,为深入研究裂解酶的理化性质和作用机制奠定了基础。

Listeria monocytogenes phage endolysin cell- wall binding domain（ CBD） can specifically recognize Listeria cells.The amplified CBDP40 gene was inserted into vector p ET32a（ ＋） and introduced into Escherichia coli expression system in order to obtain the recombinant strain. The recombinant cells were induced with IPTG and the recombinant protein was analysed for its activity.An evident induced protein band at 38 ku was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（ SDS- PAGE） after 0.1 mmol / L IPTG induction,which was in accord with the expected molecular weight. Recombinant protein CBDP40 was obtained in the supernatant after cell sonification and purified by nickel- chelating affinity chromatography with the yield of0.908 mg / m L in the elute. The results of cell binding assay and indirect ELISA showed that CBDP40 had biological activity with the function of Listeria identification. Recombinant expression,purification and activity determination of CBD may lay the foundation for further studying the physico- chemical properties and mechanism of endolysin.