噬菌体Bp7穿孔素holin的克隆、表达及其活性分析

Cloning,Expression and Activity Analysis of Phage Bp7 Holin

为了研究噬菌体Bp7穿孔素holin的特征及其对细菌的抑制作用,根据噬菌体Bp7的基因序列,设计引物PCR扩增穿孔素holin基因,并对其序列进行比对和分析。构建holin原核重组表达质粒pCold-holin,在大肠杆菌BL21中诱导表达,摸索最佳表达条件。亲和层析法纯化蛋白质,并对其抑菌活性进行测定。结果表明,噬菌体Bp7的holin蛋白含有一个跨膜区,属于Phage-holin-T家族;在E.coli BL21表达的重组holin蛋白以可溶性形式存在。表达菌E.coli BL21浊度测定试验结果表明holin蛋白的表达在4h内能够显著抑制表达菌E.coli BL21的增殖,活菌数减少,菌体出现凝集现象。研究结果提示噬菌体Bp7的holin蛋白具有抑菌活性,这将为进一步研究穿孔素的抑菌机制奠定基础。

To identify the sequence character and antibacterial activity of holin from phage Bp7, holin gene of phage Bp7 was amplified by PCR and amino acid sequence was compared and analyzed. Recombined plasmid pCold-holin was constructed and transferred into E. coli BL21 to express holin protein. The best induction conditions were explored. The protein was purified by affinity chromatography and its antibacterial activity was determined. The results showed that the holin of phage Bp7 contained a transmembrane region and belonged to Phage-holin-T family. The soluble protein was expressed in E. coli BL21. Turbidity test of E. coli BL21 showed that the expression of holin protein could significantly inhibit the proliferation of E. coli BL21, decrease the number of cells in 4 h, also the cells showed a agglutinative form. The results indicate that holin of phage Bp7 has antibacterial activity, which is helpful to further study of the antibacterial mechanism of holin.