戊型肝炎病毒第Ⅳ基因型毒株中和抗原表位的鉴定

目的确定在我国新发现的戊型肝炎病毒（HEV）第Ⅳ基因型毒株的中和抗原表位特征及其与世界各地其他基因型HEV毒株中和抗原表位的异同。方法制备HEV第Ⅳ基因型ORF2重组衣壳蛋白p166Chn及其单克隆抗体（McAb），采用体外中和试验鉴定McAb的中和活性；通过间接ELISA和免疫印迹法测定中和性McAb与不同基因型HEVORF2编码蛋白p166的免疫反应性，并结合相加ELISA确定p166Chn中和抗原表位的分布和性质。结果获得6株稳定分泌抗-p166Chn McAb的杂交瘤细胞株。所得McAb能在体外中和HEV中国毒株（第Ⅳ基因型）对PLC/PRF／5细胞的感染性，并与来源于HEV4种不同基因型代表株的7种p166重组蛋白（p166Chn、p166Bur、p166Mor、p166Pak、p166Mex、p166Us和p166Nz）均能发生阳性反应。抗体间的相加ELISA结果为阴性。表明6株McAb识别p166上的同一个中和抗原表位。结论在我国新发现的HEV第Ⅳ基因型毒株与分布在世界各地的其他基因型毒株具有共同的中和抗原表位。

Identification and characterization of neutralizing antigen epitope of hepatitis E virus clustered in genotype Ⅳ

Objective To identify and to characterize neutralizing antigen epitopes of a newly identified Chinese strain of genotype IV of hepatitis E virus (HEV) and to compare the epitopes with those of HEV strains clustered into other three different genotypes. Methods Recombinant capsid protein p166Chn and monoclonal antibodies (McAb) against the p166Chn were prepared. Neutralizing activity of the McAb was tested by an in vitro PCR-based HEV neutralization assay. The neutralizing antigen epitopes in the genotype IV isolated in China and other strains with different genotypes were analyzed by ELBA and Western blot combined with an additive ELISA assay. Results Six hybridoma cell lines secreting specific McAb against p166Chn were obtained. The McAb could effectively neutralize the infectivity of the homogenous HEV strain to PLC/PRF/5 cells. They strongly reacted with seven different p166 recombinant proteins including p166Chn,p166Bur,p166Mor,p166Pak,p166Mex,p166Us and p166Nz, which were derived from different HEV reference strains of genotype I , II , III and IV, respectively. Moreover, the additive ELBA assay showed negative reactions of McAb. The data indicated that all the McAb recognized a common neutralizing antigen epitope on the p166 proteins. Conclusion The newly identified HEV genotype IV possesses a same neutralizing antigen epitope as that derived from HEV strains of genotype I , H and ffl worldwide.