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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。
The deletion mutant (aa384-606) of Hepatitis E Virus (HEV) open reading frame 2 (ORF2) was amplified by PCR and subcloned into the expression vector, then transformed into E.coli Induced expression, the expression of the protein named rP24. SDS-PAGE and Western blotting experiments showed that rP24 was highly expressed and had strong reactivity with the monoclonal antibody 15B2. The rP24 was purified by inclusion body washing, refolding, ion-exchange chromatography and molecular sieve chromatography. Western blotting showed that the purified rP24 could react with anti-HEV ORF2 neutralizing monoclonal antibody 8C11 and HE (Hepatitis E, HE) Strong immunoreactivity, indicating rP24 conformational neutralizing epitope, mimicking the HEV capsid protein spatial structure. The results of dynamic light scattering showed that the average radius of hydration of rP24 was 7.48 nm. The experiment of purified rP24 immunized animals showed that rP24 had strong antigenicity, the positive cycle of mice was short and the duration of antibodies was longer. The purified rP24 was used as coating antigen The detection of HE positive serum and negative serum showed that the positive detection rate of HE positive serum and negative serum of rP24 was consistent with the detection rate of Beijing Wantei anti-HEV-IgG detection kit. These experimental results demonstrate that rP24, which has better immunoreactivity and immunogenicity, is highly expressed and mimics the neutralizing epitope of the native viral capsid protein for further study of the differences between genotype I and genotype IV HEV infections The molecular mechanism of host cell differentiation lays the foundation.