摘要
目的研究戊型肝炎病毒(HEV)第Ⅳ基因型巾国株开放阅读框架(ORF)2编码蛋白p166Chn的抗原表位特征。方法制备HEV ORF2重组衣壳蛋白p166Chn的特异性单克隆抗体(McAb)．进行中和活性鉴定,并利用7种HEV不同基因型p166重组蛋白以及22种N端或C端逐步截短的p166Chn进行酶联免疫吸附试验(ELISA)检测,确定McAb所识别抗原表位的性质和位置。结果获得1株稳定分泌HEV McAb的杂交瘤细胞株,命名为B_4。该McAb不与其他基因型p166重组蛋白反应,不能中和HEV对培养细胞的感染性,也不能竞争抑制已知中和性McAb与抗原p166Chn的结合,表明McAb B_4所针对的抗原表位是HEV第Ⅳ基因型特异的非中和性抗原表位。此外,McAb B_4能与截短蛋白pN452、pN460、pN462、pN463、pN464、pN465、pN466、pN468、pN470和pN472发生阳性反应,而与pN482、pN492、pC587、pC597、pC599、pC600、pC601、pC603、pC605、pC607、pN465- C601和pN460-C605均不反应。表明其可识别的抗原表位位于472～617位氨基酸,而且N端第472～482位氨基酸以及C端第607～617位氨基酸的1个或多个氨基酸对构成该抗原表位起着重要作用。结论所发现的HEV第Ⅳ基因型特异性抗原表位是1个非中和性的构象依赖性表位,可定位于pORF2的第472～617位氨基酸。
Objective To identify and characterize antigenic epitopes on the ORF2-cncoded protein p166Chn of Hepatitis E virus （HEV） genotype Ⅳ. Methods Mouse monoclonal antibodies （McAb） against the p166Chn were prepared. Neutralizing activities of these MeAbs were examined by an in vitro PCR-based HEV neutralization assay. The antigen specificity of these McAbs was examined against 7 recomhinant p166 proteins from different HEV genotypes and subtypes. The epitope mapping was carried out against 22 truncated forms of the recombinant p166Chn protein in different length. Results One McAb, named B4 and specific for p166Chn, was obtained. The McAb B4 could not react to the recombinant proteins derived from different genotypes of HEV other than that from genotype Ⅳ . McAb B4 could neither neutralize HEV infection to targeted cells nor competitively hlock the binding of a known neutralizing McAb 5G5 to the recombinant p166Chn. Detailed epitope mapping was performed which showed reactivity of the MeAb B4 to the truncated proteins of pN452, pN460, pN462, pN463, pN464, pN465, pN466, pN468, pN470 and pN472, but not to pN482, pN492, pC587, pC597, pC599, pC600, pC601, pC603, pC605, pC607, pN465-C601 and pN460-C605. One or more anfino acids from both the regions of 472 to 482 and 607 to 617 contributed to the recognition by McAb 134. Conclusion A genotype Ⅳ specific McAb B4 is produced which recognizes a conformation-dependent and non-neutralizing epitope on HEV. The epitope is located at positions between amino acids 472 and 617 of the ORF2 of HEV genotype Ⅳ.
出处
《微生物与感染》
2006年第3期149-152,共4页
Journal of Microbes and Infections
基金
国家自然科学基金(30271231)
东南大学优秀博士生学位论文基金
关键词
戊型肝炎病毒
抗原表位
单克降抗体
基因型
Hepatitis E virus
Antigenic epitope
Monoclonal antibody
Genotype
