伪狂犬病毒感染所需关键宿主因子的筛选与功能研究

伪狂犬病毒(PRV)是一种危害猪等多种动物的DNA疱疹病毒，研究PRV所需的关键宿主因子对于预防和治疗PRV感染具有重要意义。前期本项目组构建了稳定表达Cas9的IPEC-J2-Cas9细胞系，建立了猪基因组sgRNA慢病毒文库及猪基因编辑技术，并且发现PRV感染IPEC-J2-Cas9后可致细胞全部死亡。在此基础上，本项目拟利用全基因组CRISPR-Cas9敲除文库技术，筛选PRV感染后的存活细胞并结合sgRNA测序分析，获得PRV感染所需宿主因子的候选基因；然后通过测定细胞活性和病毒滴度及qPCR、透射电镜分析，鉴定候选基因敲除对细胞存活、PRV复制和入胞的影响，确定PRV复制、入胞所需关键宿主基因，进而利用荧光标记、激光共聚焦显微镜和流式细胞仪等研究目标基因在PRV吸附、膜融合和内吞过程中的功能。预期结果将揭示PRV早期感染的分子机制并为抗病毒药物设计和猪抗性遗传改良奠定基础。

Pseudorabies virus (PRV) is a DNA herpes-virus that infects pigs and many other animals. The study of key host factors essential for PRV is of great significance for the prevention and treatment of PRV infection.So far, the host factors essential for PRV infection remain poorly characterized. Recently, we have generated a stable Cas9 expressing line (IPEC-J2-Cas9) and a porcine genome-wide lentivirus sgRNA library, and successfully performed genome editing in porcine cell. Besides,we have found that the mortality rate of PRV-infected IPEC-J2-cas9 cells was up to 100%. Based on our previous work, this project intends to take advantage of the prepared lentivirus sgRNA library to perform high-throughput gene knockouts screening in the IPEC-J2-Cas9 cell line with PRV infection, combining with sgRNA sequencing and bioinformatics analysis to obtain the candidate genes of host factors required for PRV infection.Then analyze the effects of single candidate gene knockout on cell survival, PRV replication and cell entry by measuring cell viability and virus titer and qPCR,electron microscopy analysis to identify the key host genes required for PRV replication and entry. And then use fluorescent labeling, laser confocal microscopy and flow cytometry to further study the function of the target gene(s) in the process of attachment, membrane fusion and/or endocytosis during PRV entry. It is expected that the results will reveal the molecular mechanism of early PRV infection and make great basis for antiviral drug design and genetic improvement of pig resistance to PRV.