戊型肝炎病毒IV型衣壳蛋白缺失突变体原核表达与性质

Prokaryotic expression and characterization of truncated mutant capsid protein of genotype IV hepatitis E virus

摘要:

采用PCR技术扩增基因IV型HEV (Hepatitis E Virus，HEV) 开放阅读框2 (Open Reading Frame 2，ORF2) 的缺失突变体 (aa384-606)，亚克隆到表达载体后，转化到大肠杆菌中进行诱导表达，表达蛋白命名为rP24。SDS-PAGE和免疫印迹实验表明，rP24获得了高效表达，且和单克隆抗体15B2具有强的反应活性。rP24经过包涵体洗涤、溶解复性、离子交换层析和分子筛层析纯化后，免疫印迹实验表明，纯化rP24能与抗HEV ORF2中和单克隆抗体8C11以及HE (Hepatitis E，HE) 阳性血清发生很强的免疫反应性，说明rP24上具有构象型中和表位，模拟了HEV衣壳蛋白的空间结构。动态光散色测量结果表明，rP24的平均水化半径为7.48 nm；纯化rP24免疫动物实验表明，rP24具有强的抗原性，小鼠阳转周期短，抗体持续时间长；纯化rP24作为包被抗原检测HE阳性血清和阴性血清，结果显示rP24对HE阳性血清和阴性血清检出率与北京万泰公司的抗HEV-IgG检测试剂盒的检出率一致。这些实验结果说明，具有较好免疫反应性和免疫原性的rP24获得了高效表达，该蛋白模拟了天然病毒衣壳蛋白的中和表位，为进一步研究基因I型和基因IV型HEV感染不同宿主细胞差异的分子机制奠定了基础。

Abstract:

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.