CD163基因敲除iPAMs的构建及其感染PRRSV的特征分析  

Construction and PRRSV Infection Characteristic Analysis of CD 163 Gene Knockout iPAMs

作　　者：董泽霞 林鑫 周期律 王楠[2] 黄雷 刘志国[2] 冯政 牟玉莲[2] DONG Zexia;LIN Xin;ZHOU Qilyu;WANG Nan;HUANG Lei;LIU Zhiguo;FENG Zheng;MU Yulian(School of Life Science and Engineering,Foshan University,Foshan 528225,China;Institute of Animal Science of CAAS,Beijing 100193,China;College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392,China;Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,China)

机构地区：[1]佛山大学生命科学与工程学院,佛山528225 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]天津农学院动物科学与动物医学学院,天津300392 [4]中国农业科学院农业基因组研究所,深圳518120

出　　处：《中国畜牧兽医》2024年第8期3471-3483,共13页China Animal Husbandry & Veterinary Medicine

基　　金：天津市科技计划项目(22ZXZYSN00030);抗病猪新品种设计与培育(2023ZD04043);中国农业科学院科技创新工程(ASTIP-IAS05)。

摘　　要：[目的]获得分化抗原163(cluster of differentiation 163,CD163)基因敲除的永生化猪肺泡巨噬细胞系(immortalized porcine alveolar macrophages,iPAMs),并从细胞水平研究CD163蛋白在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)入侵过程中的作用。[方法]将靶向CD 163基因第7外显子的sgRNA质粒(pX330-sgCD163)转染至iPAMs,转染48 h后通过有限稀释法筛选单克隆细胞并进行基因型鉴定,通过Western blotting和脱靶分析来筛选CD 163基因敲除的iPAMs;通过实时荧光定量PCR、Western blotting、间接免疫荧光试验(indirect immunofluorescence assay,IFA)和半数细胞培养物感染量(50%tissue culture infective dose,TCID 50)等试验分析CD 163基因敲除的iPAMs感染PRRSV的特征。[结果]测序结果表明,100株单克隆细胞中有1株CD 163双等位基因敲除的iPAMs,敲除效率为1%;Western blotting结果显示,CD 163基因敲除的iPAMs中检测不到CD163蛋白的表达,且在预测的脱靶位点未检测到脱靶效应。实时荧光定量PCR结果表明,与野生型iPAMs组相比,CD 163基因敲除的iPAMs组病毒拷贝数极显著降低(P<0.01);Western blotting和IFA结果表明,在接毒24 h后的CD 163基因敲除的iPAMs组中未检测到PRRSV-N蛋白的表达;TCID 50试验结果同样显示,在CD 163基因敲除的iPAMs组中未观察到细胞病变。[结论]本研究利用CRISPR/Cas9编辑系统成功构建了CD 163基因敲除的iPAMs,该细胞系可以完全抵抗PRRSV感染。该细胞系的建立为后续研究PRRSV与CD163蛋白的互作机制提供了新型试验材料。[Objective]The aim of this study was to obtain immortalized porcine alveolar macrophages(iPAMs)with cluster of differentiation 163(CD163)gene knockout,and investigate the role of CD163 protein in the invasion process of Porcine respiratory and reproductive syndrome virus(PRRSV)at the cellular level.[Method]The sgRNA plasmid(pX330-sgCD163)targeting the exon 7 of CD 163 gene was transfected into iPAMs,followed by monoclonal cell selection and genotype identification using a limited dilution method after 48 h of transfection.CD 163 gene knockout iPAMs were screened by Western blotting and off-target analysis.The PRRSV infection characteristics of CD 163 gene knockout iPAMs were analyzed by Real-time quantitative PCR,Western blotting,indirect immunofluorescence assay(IFA)and 50%tissue culture infective dose(TCID 50).[Result]Sequencing results showed that one of the 100 monoclonal cells was CD 163 gene knockout iPAMs,with an efficiency of 1%.Western blotting results showed that there was no expression of CD163 protein in CD 163 gene knockout iPAMs,and no off-target effects were detected at the predicted off-target sites.Real-time quantitative PCR results showed that compared with wild-type iPAMs group,the viral copy number of CD 163 gene knockout iPAMs group was extremely significantly decreased(P<0.01).The results of Western blotting and IFA showed that there was no expression of PRRSV-N protein in CD 163 gene knockout iPAMs group after 24 h of post-infection.TCID 50 assays also revealed that there was no cytopathic lesions in CD 163 gene knockout iPAMs group.[Conclusion]This study successfully constructed CD 163 gene knockout iPAMs using CRISPR/Cas9 editing system,which completely resisted PRRSV infection.The establishment of this cell line provided novel research materials for investigating the interaction mechanism between PRRSV and CD163 protein in future studies.

关 键 词：CRISPR/Cas9 CD 163基因 永生化猪肺泡巨噬细胞系(iPAMs) 猪繁殖与呼吸综合征病毒(PRRSV) 

分 类 号：S852.659.6[农业科学—基础兽医学]