猪、兔和禽戊型肝炎病毒纳米抗体抗病毒感染机制研究

Hepatitis E virus (HEV) is a zoonotic virus and swine and rabbit are natural hosts. HEV infected approximately 20 million people worldwide and caused 60,000 deaths every year. For prevention and control of HEV infection in population， it is important to control HEV infection in animals. In our previous study, nanobodies that infect with the capsid proteins of swine, rabbit and avian HEVs were produced by phage display technology. In this project, the neutralizing activities of these nanobodies will be determined by virus neutralization test both in vitro and in vivo. Furthermore, the crystal structure of swine, rabbit and avian HEV capsid protein-nanobody complex will be analysed with single crystal X-ray diffraction technique to define the molecular structure of antiviral activities. Finally, the role of these molecular structures in virus infection of host cells will be characterized by reverse genetics method. Overall, the project is to elucidate the anti-swine, rabbit and avian HEV mechanism of neutralization nanobodies and confirm the role of key amino acids of HEV capsid proteins for virus infection of host cells, and provide new antiviral targets for the designing of anti-HEV drugs.

戊型肝炎病毒（Hepatitis E virus，HEV）是一种人畜共感染病毒，猪和兔等动物是其自然储存宿主。全球每年约有2000万人感染HEV并有6万人死亡，从源头阻断动物源性HEV的感染对人群中HEV的防控具有重要意义。本项目以同时结合猪、兔和禽HEV衣壳蛋白的纳米抗体为材料，通过体内外的病毒中和实验，验证纳米抗体抗猪、兔和禽HEV中和活性；然后利用单晶X射线衍射技术，解析具有中和活性的纳米抗体与HEV衣壳蛋白形成的互作结构，明确其分子结构基础；再利用反向遗传操作技术明确HEV衣壳蛋白关键氨基酸在病毒感染宿主细胞中的作用。本课题旨在分子水平上阐明纳米抗体中和猪、兔和禽HEV病毒的分子机制，为研制新型抗HEV药物提供新的靶标。

HEV是一种人畜共感染性病毒，猪和兔是其主要自然储存宿主。全球每年约有2000万人感染HEV并有6万人死亡，从源头阻断动物源性HEV的感染对人群中HEV的防控具有重要意义。本项目以同时结合猪、兔和人HEV衣壳蛋白的纳米抗体为材料，通过体内外的病毒中和实验，截短蛋白寻找互作区域，反向遗传操作技术及质谱分析，旨在分子水平上阐明纳米抗体中和猪、兔和人HEV病毒的分子机制。结果显示筛选的4株纳米抗体（nb12, nb47, nb60和nb80）均具有良好的抑制兔HEV-VLP239蛋白或CHN-SX-rHEV吸附HepG2细胞；而其中nb12和nb80可以有效抑制CHN-SX-rHEV在兔子体内增殖；2株纳米抗体结合兔HEV-VLP239蛋白的区域位于aa442-451；基于这些氨基酸的突变病毒不能感染HepG2细胞；质谱结果中筛选到CDC42差异蛋白；进一步验证兔HEV-VLP239蛋白与CDC42互作，提示CDC42可能是HEV感染宿主细胞得重要因子。根据这些结果我们推测CHN-SX-rHEV与nb12结合，阻碍了其与CDC42互作，从而发挥抑制病毒增殖的作用。本研究从分子水平上阐明中和纳米抗体抗猪、兔和人HEV病毒的分子机制，并明确HEV衣壳蛋白某些关键氨基酸在病毒感染宿主细胞中的作用，为研制新型抗HEV药物提供新的靶标。