戊型肝炎病毒（Hepatitis E virus, HEV）感染所致的戊型肝炎严重危害人类健康和畜禽养殖业健康发展。然而，病毒释放机制的不清楚严重阻碍HEV高效增殖体系的建立。前期研究发现，成熟的ORF3蛋白与病毒释放密切相关，本项目将以课题组前期筛选的与猪、兔和禽HEV ORF3蛋白互作的宿主内质网蛋白TXNDC5为研究的靶蛋白，利用RNA干扰，缺氧培养和CRISPR/Cas9等技术改变或敲除宿主细胞TXNDC5蛋白的表达，随后转染病毒感染性克隆分析该蛋白在病毒释放中的作用；然后通过Pull down和丙氨酸扫描等技术确认两者互作并解析互作的分子基础，利用对角线电泳或晶体结构解析对成熟ORF3蛋白中二硫键进行定位分析；最终通过共表达两个蛋白和构建感染性克隆突变体等技术揭示TXNDC5蛋白促进病毒ORF3蛋白成熟并参与调控猪、兔和禽HEV释放的机制，为病毒释放分子机制的深入研究提供新思路。

Hepatitis E virus (HEV), as a fecal-oral transmitted viral pathogen, was thought to be a public health problem only for the developing countries for a long time. However, discovery of HEV in swine leads researchers to realize that HEV is zoonotic pathogen and can be transmitted to humans. To date, swine and rabbit were recognized as the reservoir of HEV. In addition, the diseases also cause seriously economic loss and destroy the healthy progress in pig, rabbit and poultry industries in China. However, because of the absence of high effective cell culture system, it seriously hinders the prevention and pathogenesis of the virus. Induced mutations and deletions have revealed the importance of ORF3 for HEV infectivity in vivo, especially for virus release. Previously, we identified the binding between thioredoxin domain-containing 5 (TXNDC5) and HEV ORF3 protein. TXNDC5 is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum. In this project, the function of TXNDC5 protein in the release phase of swine, rabbit and avian HEV will be analyzed using RNA interfering, hypoxia culture and CRISP/Cas9 assays. Subsequently, the key amino acids of the interaction between TXNDC5 and HEV ORF3 proteins will be determined using truncated protein expression, mutated key amino acids, GST-Pull down and Co-IP. Meanwhile, the function of interaction between the two proteins in the release phase of HEV will be also analyzed. In addition, the arranging disulfide bonds of ORF3 protein involving the viral release will be determined by non-reducing SDS-PAGE and diagonal electrophoresis assays. Finally, the mechanism of the TXNDC5 protein involving the release phase of swine, rabbit and avian HEV will be documented by different mutated proteins and infectious cDNA clones. The expected results will provided the new sights of HEV replication and developing the high effective culture system in vitro.