基于CRISPR筛选发现的敲除猪CALR基因抑制乙型脑炎病毒增殖的抗病毒机制研究

基于CRISPR/Cas9技术及系统生物学方法是目前动物抗病性遗传基础研究新手段。流行性乙型脑炎是一种严重危害养猪业的人畜共患病，由乙型脑炎病毒（JEV）感染引起。然而，由于对JEV致病机理仍不完全清楚，限制了对猪相关抗病性分子遗传基础的认识。为阐明JEV与宿主互作的分子机制，申请人利用前期建立的猪全基因组CRISPR/Cas9敲除文库，成功筛选到多个与JEV感染相关的宿主因子，并证实敲除CALR基因能显著抑制JEV增殖，但其作用机理未知。本项目拟在前期工作基础上，进一步采用基因敲除和过表达策略，并联合转录组学、定量磷酸化蛋白质组学与蛋白互作组学等技术，阐明CALR基因及其互作蛋白、以及相关的Ca（2+）信号通路在JEV感染中的作用。通过遗传学和病毒学交叉研究，项目将揭示敲除CALR基因抑制JEV增殖的抗病毒作用机制，这不仅将深化对JEV致病机理的认识，还可为猪抗病性的遗传基础提供新理论。

CRISPR/Cas9 technology and systems biology method are the new strategies for the basic genetics research of animal disease resistance. Japanese encephalitis is a zoonotic disease that seriously endangers the pig industry and is caused by Japanese encephalitis virus (JEV) infection. However, the pathogenesis of JEV is still not fully understood, which also limits the understanding of the molecules genetic basis of relevant disease resistance in pigs. To elucidate the molecular mechanism of JEV-host interactions, using a porcine genome-scale CRISPR/Cas9 knockout screening technique, applicant have already successfully found multiple host factors associated with JEV infection. It was further confirmed by experiments that knockout of the CALR gene can significantly inhibit JEV proliferation. However, the molecular mechanism of this gene regulating the proliferation of JEV is still unknown. Based on the previous research work, this project intends to further use CRISPR/Cas9-meidiated knockout and overexpression strategies, and combine transcriptomics, quantitative phosphorylation proteomics, immunoprecipitation and mass spectrometry (CoIP-MS), to elucidate the roles of CALR gene and its interacting proteins, CALR-mediated Ca(2+) signaling pathways in JEV infection. By combining genetics and virology research strategies, the project will reveal the antiviral molecular mechanism of knockout CALR gene inhibition JEV proliferation, which will not only deepen the understanding of the pathogenesis of JEV, but also provide new theories on the molecular genetic basis of disease resistance in pigs.