深海嗜热噬菌体GVE2头尾连接蛋白EV8的原核表达和功能分析

Prokaryotic Expression and Functional Characterization of Head-tail Joining Protein EV8 from Thermophilic Bacteriophage GVE2

为了解头尾连接蛋白在噬菌体感染和装配中起的作用,对高温噬菌体GVE2(virulent bacteriophage ofGeobacillus sp.E263)的头尾连接蛋白EV8进行了原核表达和功能鉴定.将EV8编码序列克隆入pGEX4T-2原核表达载体,并转染大肠埃希氏菌BL21.诱导表达后用SDS-PAGE进行鉴定,显示获得相对分子质量约36kD的谷胱甘肽硫转移酶(GST-EV8)融合蛋白.Western blot检测发现EV8在GVE2感染后2 h开始表达,说明该蛋白为噬菌体晚期表达蛋白.免疫电镜定位表明头尾连接蛋白位于噬菌体头尾的连接处,为进一步研究高温噬菌体的组装和表达调控奠定了基础.

To investigate the role of head-tail joining protein in viral infection and DNA packaging,the head-tail joining protein EV8 from the deep-sea thermophilic bacteriophage GVE2 was characterized.EV8 encoding gene was cloned into prokaryotic expression vector pGEX4T-2 and transformed into E.coli BL21 for expression.The purified proteins characterized by SDS-PAGE and the 36 kD GST-EV8 protein was obtained.The Western blot results demonstrated that the EV8 gene was expressed in the 2 hours of GVE2 infection,implying that it might be a viral late gene.As revealed by immuno-electron microscopy,the gold particles were observed in the junction between the phage head and the phage tail.The result may help to reveal the mechanism of assembly and transcriptional regulation of thermophilic bacteriophage.