Xu Mingjie, Sun Lizhi, Wang Yan, Gao Shuchun, Yang Weihua, Li Meng
Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong 250013, P.R. China.
Department of Liver Disease, Jinan Infectious Disease Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Exp Ther Med. 2021 Feb;21(2):110. doi: 10.3892/etm.2020.9542. Epub 2020 Nov 27.
The hepatitis E virus (HEV) capsid protein pORF2 comprises three potential N-linked glycosylation sites. One site, N562, is located at the cell attachment and neutralizing antigenic regions. The present study performed detailed analyses of the effects of specific amino acid substitutions at position 562 in the homodimerization, glycosylation, antigenicity, immunogenicity and neutralization activities of HEV pORF2. Recombinant HEV pORF2 glycoprotein E1 (amino acids 439-617) and three mutant variants (N562L, N562C and N562K) were expressed in (. ) and SDS-PAGE, Western blot analysis, tunicamycin assay, double-antibody sandwich ELISA and PCR-based neutralization assay were performed to characterize the different constructs. All proteins were indicated to be secreted by . and formed homodimers. Tunicamycin assay revealed the glycosylated status of the wild-type protein, but the mutants were indicated to be non-glycosylated. All proteins were immunoreactive with a neutralizing monoclonal antibody but were not recognized by the antibody after denaturation into monomers. An PCR-based neutralization assay using mouse antibodies indicated efficient neutralization against N562L, whereas antibodies against N562C and N562K were revealed to be non-neutralizing. Collectively, the present study indicated that specific amino acid substitutions at position 562 serve crucial roles in the activity of the HEV neutralizing epitope.
戊型肝炎病毒（HEV）衣壳蛋白pORF2包含三个潜在的N-连接糖基化位点。其中一个位点N562位于细胞附着和中和抗原区域。本研究对HEV pORF2第562位特定氨基酸取代在其同源二聚化、糖基化、抗原性、免疫原性和中和活性方面的影响进行了详细分析。重组HEV pORF2糖蛋白E1（氨基酸439 - 617）和三个突变变体（N562L、N562C和N562K）在（.）中表达，并进行了SDS-PAGE、蛋白质免疫印迹分析、衣霉素检测、双抗体夹心ELISA和基于PCR的中和试验，以表征不同构建体。所有蛋白质均显示由（.）分泌，并形成同源二聚体。衣霉素检测揭示了野生型蛋白的糖基化状态，但突变体显示为非糖基化。所有蛋白质与一种中和单克隆抗体具有免疫反应性，但变性为单体后不被该抗体识别。使用小鼠抗体进行的基于PCR的中和试验表明对N562L有高效中和作用，而针对N562C和N562K的抗体显示无中和作用。总体而言，本研究表明第562位特定氨基酸取代在HEV中和表位的活性中起关键作用。
