DF-1细胞中TLR7编码基因的敲除及其抗FAdV-4感染的天然免疫应答  

Knock-out of TLR7 coding sequence in DF-1 cells and its anti-FAdV-4 innate immune response

作　　者：恽君雯 陈丽[3] 徐悦[3] 鲍熹[3] 冯磊[3,4,5] 高崧 YUN Junwen;CHEN Li;XU Yue;BAO Xi;FENG Lei;GAO Song(Productivity Centre of Jiangsu Province,Nanjing 210042,China;Yangzhou University College of Veterinary Medicine,Yangzhou 225009,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;School of Pharmacy,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏省生产力促进中心,江苏南京210042 [2]扬州大学兽医学院,江苏扬州225009 [3]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [4]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [5]江苏大学药学院,江苏镇江212013

出　　处：《畜牧与兽医》2023年第2期80-90,共11页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发专项(2022YFD1800800);江苏省重点研发计划(BE2022787)。

摘　　要：旨在构建高质量的禽源细胞系,应用CRISPR/Cas9技术实现鸡胚成纤维细胞(DF-1)的Toll样受体7(TLR7)编码基因的敲除,构建稳定的细胞培养禽腺病毒4型(FAdV-4)增殖体系。采用荧光定量PCR的方法对FAdV-4感染DF-1细胞后先天性免疫信号通路中效应分子进行检测,选择转录水平显著上调的基因TLR7作为研究对象;构建sgRNA载体与Cas9表达载体共转染DF-1细胞,经绿色荧光蛋白(GFP)阳性特征分选单克隆,PCR验证及增毒试验筛选后获得TLR7编码序列敲除的DF-1-TLR7-KO#3单克隆细胞系,命名为DF-1-TLR7-KO细胞。对敲除前后DF-1细胞的天然免疫系统对FAdV-4感染的拮抗效应和病毒在不同细胞中增殖效率的差异进行了研究。结果表明:成功构建的TLR7编码序列敲除的DF-1细胞系,与原始细胞相比病毒增殖能力提高。试验对细胞的天然免疫系统与FAdV-4感染的互作机理进行了初步探索,为更好地研究宿主对病毒入侵的应答机制,提高疫苗生产效能提供了研究基础和生物材料储备。In order to construct highly qualified avian cell lines,the TLR7 coding gene of DF-1 cells was knocked out by the CRISPR/Cas9 system.And stable cell-derived proliferation of fowl adenovirus 4(FAdV-4)was constructed.Firstly,the key genes of the innate immune system in the FAdV-4 infected DF-1 cells were detected by qPCR,and the genes with significant difference in transcriptional level were selected.TLR7 was selected as the target gene to construct an sgRNA vector,which was co-transfected with the Cas9 expression vector into DF-1 cells.Then,single cell clones were sorted and cultured according to their GFP positive characteristics.After PCR verification and virus replication screening,DF-1-TLR7-KO#3 single cell clones were selected and named DF-1-TLR7-KO cells.These cell clones showed the best potence of virus proliferation.Next,the antagonistic mechanism of the innate immune system against FAdV-4 infection and the difference of virus proliferation efficiency between pre-modified and post-modified DF-1 cells were investigated.Thus,a DF-1 cell line with the TLR7 gene knocked-out was successfully constructed,and the virus proliferation ability of the cell line was improved.Finally,the mechanism of the interaction between FAdV-4 infection and the innate immune system of avian cells was also explored in this research.The present study provides a basis for studying the response mechanism to virus invasion in cells,and supplies a material reserve for improving vaccine production efficiency.

关 键 词：免疫应答 DF-1细胞 禽腺病毒4型 CRISPR/Cas9 TOLL样受体7 

分 类 号：S852.6[农业科学—基础兽医学]