Effects of ATP6V0A1，IL20RB and STX10 gene on proliferation of PRRSV and PEDV
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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。
Abstract:
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-Cas9 cells 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 canplay important roles in the proliferation ofPRRSV and PEDV.