戊型肝炎病毒衣壳蛋白中和表位间的构象诱导

Induced Conformational Changes between the Neutralizing Epitopes of Hepatitis E Virus Capsid Protein

重组蛋白NE2包含了戊型肝炎病毒(HEV)衣壳蛋白(pORF2)的aa394～606片段。在NE2上已鉴定出了2个HEV中和表位,并获得了3个识别中和表位的单克隆抗体(MAb)8C11、13D8和8H3。这3个MAb间的交叉阻断ELISA实验发现,8C11和13D8可以彼此完全阻断,8H3对8C11和13D8均不能阻断,而8C11非但不能阻断8H3,反而显著增强了8H3与抗原的结合。用生物传感器进行的抗体与抗原结合的动力学分析也证实了这一现象。这些结果提示,在NE2上8H3表位区域受到抗原上某些结构的掩盖,而8C11与NE2的结合引起了抗原空间结构的改变,导致了掩盖8H3表位的结构的去除和8H3表位的充分暴露。免疫捕获RT PCR发现,8C11同样可以显著增强8H3对天然HEV病毒的捕获能力,提示这种结合诱导的衣壳蛋白空间构象改变在天然HEV病毒颗粒上同样存在。

Recombinant antigen NE2,comprising aa 394-606 of the capsid protein of hepatitis E virus(HEV), is believed to model the important structural feature of the virus particle. Recently,two neutralizing epitopes of HEV had been identified on NE2 by three neutralization monoclonal antibodies(MAbs),8C11,13D8 and 8H3.Cross-blocking assay showed that 8C11 and 13D8 blocked each other almost completely,but 8H3 could not block these two monoclonal antibodies.However,instead of blocking,8C11 enhanced significantly the binding of 8H3 with the NE2 antigen.Kinetic studies using a biosensor showed that the NE2 antigen has limited access by the native antibody but not the smaller Fab.Similarly as blocking ELISA,biosensor assay showed that the binding of 8C11 could significantly enhance the binding of antigen with native antibody 8H3 as well as 8H3 Fab.Immune capture of virus also showed similar result that pre-incubation of 8C11 with virus enhanced significantly the binding of 8H3 with the virus.These results suggested that the 8H3 epitope is partially masked on the surface of HEV virion,and the binding of 8C11 epitope site might induce the conformational changes on the capsid protein,which unmasks the 8H3 epitope site.