鸡chNHE1精准基因编辑细胞系的构建及其抗ALV-J感染的研究

Construction of Chicken chNHE 1 Gene Editing Cell Line and Analysis of Its Resistance to ALV-J Infection

旨在构建ALV-J受体分子chNHE1精准基因编辑细胞系,本研究利用荧光标记的CRISPR/Cas9系统,在DF-1细胞中将chNHE1介导ALV-J进入宿主细胞的关键氨基酸V33进行突变,W38进行缺失,同时将编码第34-37位氨基酸的密码子同义替换。通过流式细胞分选获得48株单克隆细胞系,PCR及测序分析结果显示,其中有14株单克隆细胞系的chNHE1成功发生V33突变、W38缺失以及34-37位氨基酸的密码子同义替换,基因编辑效率为29%。为了验证chNHE1基因编辑DF-1细胞系的遗传稳定性及增殖水平,对传至第25代的细胞系进行测序分析,结果显示,chNHE1基因未发生回复性突变;进一步细胞计数分析结果显示,chNHE1基因编辑细胞系增殖水平未受到影响;为了评价chNHE1基因编辑细胞系抗ALV-J感染的能力,分别利用ALV-J荧光报告病毒(ALV-J-GFP)及ALV-J原型毒株(HPRS-103)对其进行病毒感染试验,荧光观察结果及流式细胞分析结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI ALV-J-GFP的感染;进一步间接免疫荧光试验、PCR扩增试验以及病毒滴度测定试验结果显示,chNHE1基因编辑细胞系可完全抵抗0.1 MOI HPRS-103毒株及0.1 MOI JL08CH3-1毒株的感染。本研究利用荧光标记的CRISPR/Cas9系统结合流式细胞分选,成功构建了chNHE1基因编辑细胞系,其可完全抵抗ALV-J的感染,且该细胞系遗传稳定性及增殖活性良好,为建立抗ALV-J感染的新技术提供了理论支持及基因编辑靶点。

This study aimed to construct a precise gene-edited cell lines for the ALV-J receptor molecule chNHE1.In DF-1 cells,key amino acids V33 and W38 facilitating ALV-J entry into host cells via chNHE1 were mutated and deleted,respectively.Meanwhile,the codon encoding amino acids 34-37 of chNHE 1 were synonymously replaced.Forty-eight monoclonal cell lines were obtained through flow cytometry sorting,and PCR identification and sequencing analysis revealed that 14 of these cell lines had chNHE1 mutations in V33,W38 deletions,and synonymous substitution of codons at amino acids 34-37,with a gene editing efficiency of 29%.In order to verify the genetic stability and proliferation of the chNHE 1 gene-edited cell lines,sequencing analysis was performed on the cells lines that were continuously passaged for 25 generations,and the results showed no revertant mutations in the chNHE 1 gene.Further cell count analysis showed no revertant mutations in the ch NHE1 gene.Further cell count analysis showed that the proliferation level of chNHE 1 gene-edited cell lines was unaffected.In order to verify the anti ALV-J infection ability of the chNHE 1 gene-edited cell line,ALV-J fluorescent reporter virus(ALV-J-GFP)and ALV-J prototype strain(HPRS-103)were used for virus challenge experiment.The fluorescence observation and flow cytometry analysis results showed that the chNHE 1 gene-edited cell line completely resisted the infection of ALV-J fluorescent strain;Furthermore,the results of indirect immunofluorescence assays,PCR amplification assays,and virus titer determination assays showed that the chNHE 1 gene-edited cell line completely resisted the infection of HPRS-103 strain.This study utilized a fluorescence labeled CRISPR/Cas9 system combined with flow cytometry sorting to successfully construct a chNHE 1 gene-edited cell line,which showed complete resistance to ALV-J infection,and exhibited good genetic stability and proliferation activity.This provides theoretical support and gene editing targets for establishing new technologies against ALV-J infections.