戊型肝炎病毒ORF2蛋白与宿主细胞蛋白的相互作用及其生物学意义

项目名称： 戊型肝炎病毒ORF2蛋白与宿主细胞蛋白的相互作用及其生物学意义

项目编号： No.30872235

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 周乙华

作者单位： 南京大学

项目金额： 32万元

中文摘要： 在本研究中，我们首先通过表达1型和4型戊型肝炎病毒(HEV) 的ORF2多肽，对其二聚体特性和抗原性特征进行了深入研究，其二聚体特性依赖于ORF2第459至607位氨基酸残基，该区域为二聚体功能域；其氨基端或羧基端截短多肽不能形成二聚体，新发现595位异亮氨酸突变能破坏ORF2的二聚体，且使其抗原性失活。然后我们研究了ORF2蛋白和肝细胞的相互作用。流式细胞仪检测的结果验证了HEV ORF2多肽吸附肝癌细胞株Huh-7的能力，并确定了4型HEV ORF2与Huh-7细胞表面结合的最短肽段。Pull-Down实验结果显示，HEV ORF2能与Huh-7细胞HSC70结合。酵母双杂交技术结果提示，HEV ORF2功能域多肽能与肝细胞中的Cytochrome C oxidase subunit和Phosphoglucomutase 1等结合。此外，在上述研究结果的基础上，基于HEV ORF2二聚体功能域多肽能与抗-HEV抗体特异性结合而截短多肽不能与抗-HEV抗体结合这一发现，建立了可排除抗-HEV IgM假阳性的抗体检测方法，提高了戊型肝炎诊断试剂的特异性。

中文关键词： 戊肝病毒；ORF2蛋白；抗原特性；宿主细胞蛋白；相互作用

英文摘要： In this study, we first characterized the dimerization and antigenicity of the hepatitis E virus (HEV) ORF2 protein based on the purified recombinant polypeptide expressed in the bacteria. The dimerization of ORF2 protein depends upon the amino acid residues 459-607, which cover the dimerization domain. The domain polypeptide truncated at either N- or C-terminus can not form dimmers. The dimerization and antigenicity of ORF2 may be destroyed by a mutation at residue 595 (isoleucine), which is a novel finding. We then investigated the interaction of ORF2 protein and hepatocytes. The results of flow cytometry demonstrated that ORF2 polypeptide bound on Huh-7 hepatocarcinoma cells and defined the minimal region responsible for the binding. Pull-Down experiment showed that HEV ORF2 polypeptide may bind with heat shock cognate protein 70. The results of yeast two hybridization indicated that ORF2 dimerization domain interacted with cytochrome C oxidase subunit, phosphoglucomutase 1, and several other proteins in human hepatocytes. Furthermore, by the combination of high reactivity of the dimerization domain polypeptide and poor reactivity of the truncated polypeptide, we established an indirect enzyme-linked immunosorbent assay to detect anti-HEV IgM, which may exclude the nonspecific binding in the detection of anti-HEV IgM and increase the specificity of the diagnostic reagents for hepatitis E.

英文关键词： hepatitis E virus; ORF2 protein; antigenicity; host cell protein; interaction