Qiu Yilan, Wu Junwen, Qiu Guo, Li Sang, Li Ye, Liu Shengzi, Liu Rushi
Sheng Wu Gong Cheng Xue Bao. 2014 Mar;30(3):381-92.
A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.
通过聚合酶链反应（PCR）从基因型IV型戊型肝炎病毒（HEV）的cDNA中扩增出开放阅读框2（ORF2，氨基酸384 - 606）的截短突变体，亚克隆至表达质粒pTO - T7，并在大肠杆菌中表达。采用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法检测和鉴定重组蛋白，即rP24。包涵体经洗涤、变性剂溶解、透析复性、离子交换层析和凝胶层析后，利用动态光散射研究rP24的水化半径。采用蛋白质印迹法检测rP24的免疫反应性，通过小鼠免疫试验和间接酶联免疫吸附测定（ELISA）评估rP24对戊型肝炎病毒阳性和阴性血清的免疫原性及检测率。SDS - PAGE和蛋白质印迹法显示，rP24诱导后以包涵体形式高表达，对单克隆抗体（McAb）15B2具有较强的免疫反应性。rP24经多步纯化后，蛋白质印迹法表明纯化后的rP24对中和性单克隆抗体8C11和戊型肝炎病毒阳性血清也具有较强的免疫反应性，提示rP24模拟了戊型肝炎病毒衣壳蛋白的天然结构。动态光散射显示纯化后的rP24平均水化半径为7.48 nm。小鼠免疫试验表明，纯化后的rP24也具有良好的免疫原性，血清抗体由阴性转为阳性的时间很短，但抗体维持时间超过20周。间接ELISA试验显示，其检测率与抗戊型肝炎病毒IgG诊断试剂盒（万泰公司）相同。综上所述，rP24模拟了天然戊型肝炎病毒的中和表位，具有较强的免疫反应性和免疫原性。为进一步研究I型和IV型戊型肝炎病毒感染机制的差异提供了依据。
