J亚型禽白血病病毒（ALV-J）尚无可用的疫苗和有效的防治手段，本项目在前期研究基础上运用创新方法对广西三黄鸡进行基因编辑抗病育种，探索建立新的ALV-J防控技术。使用单碱基基因编辑技术对鸡胚成纤维细胞系（DF-1）中ALV-J的结合蛋白NHE1进行多个位点的定点突变，建立单克隆细胞株；包装绿色荧光标签的ALV-J，对各个细胞株攻毒，筛选和评估各突变的抗病毒效果；检测抗病毒突变对DF-1代谢、增殖、凋亡表型的影响；检测单碱基基因编辑脱靶效应。使用腺病毒载体直接注射法制备基因编辑鸡：将抗病毒突变对应的单碱基编辑元件包装至腺病毒载体，探索优化显微注射鸡胚的条件和基因编辑效率；根据优化条件注射鸡胚，孵化后鉴定阳性F0代并进行繁育。使用ALV-J标准毒株对F2代基因编辑雏鸡及阴性对照鸡攻毒，分期采样进行血清抗原ELISA检测及病理切片检测。本项目的实施将为J亚型禽白血病的防治提供新的思路。

For avian leukosis virus subgroup J (ALV-J), no vaccines and effective treatments are currently available. Based on previous research, this project will use an innovative method to produce ALV-J resistant Guangxi Sanhuang chicken. Multiple site mutations of ALV-J binding protein NHE1 in chicken embryo fibroblast (DF-1) will be performed using base editing and multiple monoclonal cell lines will be established. ALV-J with GFP tag will be packaged, and each cell line will be subjected to virus challenge to detect the virus resistance of each mutation. The effects of virus-resistant mutations on DF-1 metabolism, proliferation, and apoptotic phenotype will be examined. Base editing off-target effects will be detected. Use in vivo delivery of base editing adenovirus to produce ALV-J resistant chickens, explore optimization of microinjection conditions and gene editing efficiency of chicken embryos, and positive chicks will be identified and bred. ALV-J standard strains will be used to challenge homozygous ALV-J resistant chickens and negative control chickens. Sampling will be performed at different time points, and serum antigen ELISA detection and pathological sections will be used for detection. The implementation of this project will provide innovative technology for the prevention and treatment of avian leukosis.