Abstract

Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368–606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.

摘要

戊型肝炎病毒（HEV）是一种无包膜病毒，引起一种新出现的人畜共患疾病，严重威胁世界公众健康，尤其是孕妇。在这项研究中，衣壳蛋白 ( t ORF2-HEV)的开放阅读框 2 的截短形式 (aa 368-606) 是HEV 的主要结构蛋白，在大肠杆菌中表达。这项工作首次表征了大肠杆菌中融合的谷胱甘肽-S-转移酶标记的t ORF2 (GST- t ORF2) 和t ORF2-HEV 形式。融合蛋白通过亲和层析纯化，纯度高于90%，凝血酶消化后产率约为27%。纯化的 GST- t然后通过蛋白质印迹、使用抗 GST 抗体和 CD 光谱表征 ORF2 蛋白。GST- t ORF2 和t ORF2-HEV 蛋白显示可有效开发 ELISA 测试，以检测用重组全长 ORF2 蛋白免疫的小鼠血清中的抗 HEV IgG。在涂有 0.5、1 和 2 µg 蛋白质的板孔中，血清显示 450 nm 处的吸光度信号显着增加。用纯化的 GST- t ORF2 和t ORF2-HEV包被的 ELISA 板显示出与 HEV ELISA 相似的反应，其中用表达完整 ORF2 的重组杆状病毒感染的总昆虫细胞裂解物用作阳性对照。