猪氨基肽酶N病毒结合受体核心区对TGEV在ST细胞中复制影响的研究  

Effect of porcine aminopeptidase N virus binding receptor core region on TGEV replication in ST cell

作　　者：史鑫琪 季朝阳 陈晓彤 张子博 张鑫[2] 郭慧娟 田璐 王洪峰[3] 陈洪岩[1] 王靖飞[4] 冯力[2] 夏长友 孟庆文[1] SHI Xin-qi;JI Zhao-yang;CHEN Xiao-tong;ZHANG Zi-bo;ZHANG Xin;GUO Hui-juan;TIAN Lu;WANG Hong-feng;CHEN Hong-yan;WANG Jing-fei;FENG Li;XIA Chang-you;MENG Qing-wen(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/Innovative Team for Laboratory Animals and Comparative Medicine/National Poultry Laboratory Animal Resource Bank,Harbin 150069,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/Division of Swine Digestive System Infectious Diseases,Harbin 150069,China;Harbin Weike Biotechnology Development Company,Harbin 150069,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences/Innovative Team for Animal Pathogens and Pathomorphology,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/实验动物与比较医学创新团队/国家禽类实验动物资源库,黑龙江哈尔滨150069 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪消化道传染病创新团队,黑龙江哈尔滨150069 [3]哈尔滨维科生物技术开发公司,黑龙江哈尔滨150069 [4]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物病原与病理形态学创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第1期1-8,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2021YFF0703000、2021YFF0703100)。

摘　　要：为研究与猪传染性胃肠炎病毒(TGEV)结合的猪氨基肽酶N(pAPN)受体核心区对TGEV复制以及细胞功能的影响,本研究检索并分析pAPN与TGEV的结合位点,结果显示,敲除pAPN aa668~aa963对其酶活性无影响。因此,利用CRISPR/Cas9技术构建pAPN与TGEV结合位点敲除细胞系,经PCR和western blot鉴定结果显示,正确构建一株敲除pAPN aa668~aa963的ST细胞系,即敲除pAPN 15-21外显子,命名为KO-ST-15。采用CCK-8和细胞划痕方法检测KO-ST-15细胞的增殖和细胞迁移活性,结果显示,敲除pAPN 15-21外显子对ST细胞增殖和迁移活性均无显著影响。利用RT-qPCR和IFA检测KO-ST-15感染TGEV后病毒复制拷贝数和N蛋白的表达,结果显示,敲除pAPN 15-21外显子可极显著抑制TGEV在ST细胞系中的复制(P<0.001)。利用RT-qPCR检测KO-ST-15感染TGEV后炎症相关基因转录水平变化,结果显示,TGEV感染后KO-ST-15中的IL-6、IL-8和NLRP3(NOD-like receptor protein 3)mRNA转录水平无显著变化,但RIG-I和MDA5的转录水平显著上调(P<0.05)。上述结果首次表明,敲除pAPN15-21外显子能够极显著抑制TGEV在ST细胞中的复制,但细胞KO-ST-15的正常生理功能无变化,TGEV不能进入敲除pAPN 15-21外显子的细胞中引起炎症反应,该结果为进一步培育抗病育种猪提供参考依据和候选方案。In order to investigate the effect of the receptor core region of porcine aminopeptidase N(pAPN)that binding to porcine transmissible gastroenteritis virus(TGEV)on TGEV replication and its cellular function,the binding sites of pAPN to TGEV were summarized and analyzed,and a TGEV binding sites knockout cell line was constructed by CRISPR/Cas9 technology in this study.A ST cell line with knockout of pAPN amino acid residues 668-963,pAPN 15-21 exons,was constructed and named as KO-ST-15,which was identified by PCR and western blot.The viral replication copy number and N protein content in TGEV-infected KO-ST-15 cells were detected using fluorescence quantitative PCR and indirect immunofluorescence assay(IFA),and the results showed that knockout of pAPN exons 15-21 inhibited the replication of TGEV in KO-ST-15 cells significantly(P<0.001).The proliferation and cell migration activity of KO-ST-15 cells were detected by CCK-8 and cell scratch assay,and the results showed that knockout of pAPN 15-21 exons had no significant effect on the proliferation and migration activity of ST cells.The changes in transcript level of inflammation-related genes in TGEV-infected KO-ST-15 cells were detected using fluorescence quantitative PCR,which showed that transcription of IL-6,IL-8 and NLRP3 mRNA in KO-ST-15 cells showed no significant changes after TGEV infection,but that of RIG-I and MDA5 were upregulated.The above results showed for the first time that knockout of pAPN exons 15-21 could significantly inhibit the replication of TGEV on ST cells,and no physiological function changes in KO-ST-15 knockout cells was found.In addition,TGEV could not enter the cells with knockout of pAPN exon 15-21 to cause inflammatory response.These results provided a theoretical basis and candidate scheme for further construction of disease-resistant breeding pigs.

关 键 词：猪氨基肽酶N 猪传染性胃肠炎病毒 CRISPR/Cas9 ST细胞 

分 类 号：S852.65[农业科学—基础兽医学]