IL20RB、ATP6V0A1和STX10基因对PRRSV和PEDV的增殖作用  

Effects of ATP6V0A1,IL20RB and STX10 gene on proliferation of PRRSV and PEDV

作　　者：王金玉 上官爱哨 孙玉梅 蒋金河 刘忠柱 张淑君[1] WANG Jinyu;SHANGGUAN Aishao;SUN Yumei;JIANG Jinhe;LIU Zhongzhu;ZHANG Shujun(School of Animal Science and Technology and School of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学动物科学技术学院与动物医学院,武汉430070

出　　处：《华中农业大学学报》2022年第2期176-184,共9页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31872328,31572367)。

摘　　要：为了明确IL20RB、ATP6V0A1和STX10基因对猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)和猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的增殖作用,利用CRISPR/Cas9技术在PK15-CD163-Cas9细胞和IPEC-J2-Cas9细胞(笔者所在实验室前期制备)中分别敲除这3个基因,用PRRSV感染基因敲除的3种细胞(PK15-CD163-Cas9-ATP6V0A1、PK15-CD163-Cas9-IL20RB和PK15-CD163-Cas9-STX10),荧光定量PCR检测PRRSV的ORF7基因表达水平,分析PRRSV增殖情况;用PEDV感染基因敲除的3种细胞(IPEC-J2-Cas9-ATP6V0A1、IPEC-J2-Cas9-IL20RB和IPEC-J2-Cas9-STX10),荧光定量PCR检测PEDV的N蛋白基因表达水平,分析PEDV增殖情况。结果显示,在3个基因分别被敲除的PK15-CD163-Cas9-ATP6V0A1、PK15-CD163-Cas9-IL20RB和PK15-CD163-Cas9-STX10细胞中病毒基因ORF7表达水平均显著低于未敲除基因的细胞(PK15-CD163-Cas9-NC,对照组),在3个基因分别被敲除的IPEC-J2-Cas9-ATP6V0A1、IPEC-J2-Cas9-IL20RB和IPEC-J2-Cas9-STX10细胞中PEDV的N蛋白基因表达水平均显著低于未敲除基因的细胞(IPEC-J2-Cas9-NC,对照组),表明敲除这3个基因对PRRSV和PEDV增殖均具有显著的抑制作用,ATP6V0A1、IL20RB和STX10对PRRSV和PEDV增殖均具有重要调控作用。The three genes including ATP6V0A1,IL20RB,and STX10 were individually knocked out with CRISPR/Cas9 technology(previously established in author’s laboratory)in PK15-CD163-Cas9cells and IPEC-J2-Cas9 cells to study the effects of three genes on the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)and porcine epidemic diarrhea virus(PEDV).The cells with knocked-gene(PK15-CD163-Cas9-ATP6V0A1,PK15-CD163-Cas9-IL20RB and PK15-CD163-Cas9-STX10)were infected with PRRSV.The expression of the ORF7 of PRRSV was analyzed with fluorescence quantitative PCR to detect the proliferation of PRRSV.Meanwhile,the cells with knocked-gene(IPEC-J2-Cas9-ATP6V0A1,IPEC-J2-Cas9-IL20RB and IPEC-J2-Cas9-STX10)were infected with PEDV.The expression of N protein gene of PEDV and the proliferation of PEDV were detected.The results showed that the expression level of ORF7 in the cells with knocked-gene was significantly lower than that of the wildtype cells(PK15-CD163-Cas9-NC,control group).The expression of N protein gene was significantly reduced in the cells with knocked-gene,compared with the wildtype cells(IPEC-J2-Cas9-NC,control group).It is indicated that individually knocking out of the three genes significantly inhibits the proliferation of PRRSV and PEDV.ATP6V0A1,IL20RB,and STX10 can play important roles in the proliferation of PRRSV and PEDV.

关 键 词：基因敲除 CRISPR/Cas9技术 猪繁殖与呼吸综合征病毒 猪流行性腹泻病毒 

分 类 号：S855.3[农业科学—临床兽医学]