含半胱氨酸的天冬氨酸蛋白水解酶(caspase-1)对猪伪狂犬病毒复制的影响

Effect of Caspase-1 on Replication of the Porcine Pseudorabies Virus

猪伪狂犬病是伪狂犬病毒(Pseudorabies virus,PRV)感染引起的一种烈性接触性传染病,其感染宿主会触发机体先天免疫应答,引起I型干扰素(Type I interferon,IFN-1)和炎性细胞因子等细胞因子的产生,为研究可诱导产生炎性细胞因子的含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase 1,caspase-1)的基因敲除对PRV复制的影响,本试验利用近年来发展迅速的一项规律性短重复回文序列簇/Cas9核酸酶(Clustered regulatory interspaced short palindromic repeat/CRISPR associated system 9,CRISPR/Cas9)基因定点修饰技术构建猪肾上皮细胞(Porcine kidney epithelial cells,PK15)caspase-1基因稳定敲除细胞系,并通过T7核酸酶检测敲除效率;细胞毒性(Cell counting kit-8,CCK-8)试剂盒检测PK15敲除caspase-1增殖影响;采用流式细胞术检测PRV-GFP感染PK15以及PK15-caspase-1^(-/-)的增殖差异;实时荧光定量PCR(Real-time quantitative PCR,RT-PCR)检测PRV-gB、TK及白细胞介素1β(Interleukin-1β,IL-1β)、IFN-β、干扰素刺激基因(Interferon-stimulated genes 20,ISG20)mRNA的表达;Western Blot检测PRV-gB蛋白表达;滴度测定检测子代病毒滴度。结果表明,2对特异性单链引导RNA(Single guide RNA,sgRNA)均能对caspase-1进行基因编辑,但经T7核酸酶酶切进行基因编辑效率分析结果表明sgRNA2的基因编辑效率较高;CCK-8试剂盒检测细胞活力结果表明caspase-1基因敲除对PK15以及PK15-caspase-1^(-/-)细胞活力无影响(P>0.05);流式细胞仪检测结果表明PRV-GFP在PK15-caspase-1^(-/-)中的增殖显著低于PK15细胞(P<0.05);定量RT-PCR结果表明PRV-gB、TK基因在PK15-caspase-1^(-/-)的mRNA表达显著低于PK15细胞(gB:P<0.05,TK:P<0.05),而IFN-β、ISG20基因在PK15-caspase-1^(-/-)的mRNA表达显著高于PK15细胞(gB:P<0.05,TK:P<0.05);Western Blot结果表明,PRV的gB蛋白在PK15-caspase-1^(-/-)的表达显著低于PK15细胞(P<0.05);滴度测定结果表明,敲除caspase-1能够抑制PRV子代病毒的增殖。以上结果均表明caspase-1基因敲除可抑制PRV在PK15细胞中复制。

Porcine pseudorabies is a severe contagious disease caused by pseudorabies virus(PRV)infection.The host of pseudorabies infection triggers the innate immune response,which leads to the production of cytokines such as type I interferon(IFN)and inflammatory cytokines.We wished to study the effect of knockout of caspase-1(which can induce secretion of proinflammatory cytokines)on PRV replication.A rapidly developed CRISPR/Cas9 gene-site modification method was used to construct porcine renal epithelial cell line(PK15)with caspase-1 gene stable knockout cell line.The knockout efficiency was detected by digestion of T7nuclease.The CCK-8 kit was used to detect the effect of proliferation of PK15 cells after caspase-1 knockout.Flow cytometry was employed to detect the difference in proliferation of PK15 and PK15-caspase-1^(-/-)cells infected with PRV-GFP.Quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to measure expression of PRVgB,TK,interleukin(IL)-1β,IFN-βand ISG20 gene mRNA.Expression of PRV gB protein was measured by Western Blot,and the titer of the progeny virus was detected by titer assays.Both pairs of specific sgRNA could edit caspase-1,but the gene-editing efficiency of sgRNA2 was high according to digestion of T7 nuclease.The CCK-8 kit showed that caspase-1 knockout had no effect on the viability of PK15and PK15-caspase-1^(-/-)cells(P>0.05).Flow cytometry showed that proliferation of PRV-GFP in PK15-caspase-1^(-/-)cells was significantly lower than that in PK15 cells(P<0.05).qRT-PCR showed that mRNA expression of gB and TK of the PRV in PK15-caspase-1^(-/-)cells were significantly lower than that in PK15 cells(gB:P<0.05,TK:P<0.05),whereas mRNA expression of IFN-βand ISG20 in PK15-caspase-1^(-/-)cells was significantly higher than that in PK15 cells(IFN-β:P<0.05,ISG20:P<0.05).Western Blot showed that expression of gB protein in the PRV was significantly lower than that in PK15 cells(P<0.05).The titer assay showed that caspase-1 knockout could inhibit proliferation of the PRV progeny virus.These results suggest that caspase-1 knockout can inhibit PRV replication in PK15 cells.