【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。更多还原

【Abstract】 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.更多还原