凋亡相关斑点样蛋白敲除PK-15细胞系的建立及对PRV感染的影响

Establishment of Apoptosis Associated Dot Like Protein Knockout PK-15 Cell Line and Its Effect on Porcine Pseudorabies Virus Infection

本试验旨在研究凋亡相关斑点样蛋白(ASC)的基因敲除对猪伪狂犬病病毒(PRV)感染PK-15细胞的影响。以慢病毒介导的CRISPR/Cas9基因编辑技术,构建猪肾上皮细胞(PK-15)ASC基因稳定敲除细胞系,通过T7核酸酶检测靶基因的敲除效率;CCK-8试剂盒检测PK-15敲除ASC基因对细胞增殖的影响;采用流式细胞术检测PK-15以及PK15-ASC-/-细胞感染PRV-GFP病毒的增殖差异;RT-PCR检测PRV-gB及IFN-β、ISG15、IL^-1βmRNA表达水平;Western blot检测PRV gE蛋白的表达;TCID50检测子代病毒感染力的滴定。结果表明:两对特异性sgRNA均能对ASC进行基因编辑,但经过T7核酸酶酶切检测进行基因编辑效率分析,结果表明,sgRNA2的基因编辑效率最高;CCK-8试剂盒检测细胞活力结果表明,PK-15以及PK15-ASC-/-细胞活力无影响;流式细胞仪检测结果表明PRV-GFP在PK15-ASC-/-细胞中的增殖显著低于PK-15细胞;定量RT-PCR结果表明,PRV-gB基因在PK15-ASC-/-细胞中的转录水平显著低于PK-15细胞,而IFN-β、ISG15基因在PK15-ASC-/-细胞中的转录水平显著高于PK-15细胞;Western blot结果表明,PRV的gE蛋白在PK15-ASC-/-细胞中的表达显著低于PK-15细胞;滴度测定结果表明敲除ASC基因能够显著抑制子代病毒的增殖能力。本研究成功构建了PK15-ASC-/-细胞系,并通过PRV感染试验证实敲除ASC基因可以抑制PRV在PK-15细胞中的增殖。

This study aimed to investigate the effect of ASC gene knockout on the infection of pseudorabies virus(PRV)in PK-15 cells.The CRISPR/Cas9 gene-editing technology mediated by lentivirus was applied to knock out the ASC gene of porcine renal epithelial cell(PK-15),and the efficiency was detected with T7 endonuclease.Flow cytometry was applied to detect the difference of PRV-GFP proliferation between in PK-15 and PK15-ASC-/-cells.The transcription levels of PRV-gB,IL^-1β,IFN-β,ISG15 genes and the expression of PRV-gE protein were tested with RT-PCR and Western blot,the titer of progeny PRV was determined with TCID50 method.The results showed that specific sgRNA could carry out efficient gene editing for ASC gene,and the activity of PK15-ASC-/-cells was not significantly affected.After infection,the proliferation of PRV-GFP in PK15-ASC-/-cells was significantly inhibited compared with that in PK-15 cells.Besides,both the transcription level of PRV-gB gene and the expression level of PRV-gE protein in PK15-ASC-/-cells were also lower than that in PK-15 cells.Nevertheless,the transcription level of IFN-βand ISG15 gene presented the opposite results.The virus titer test showed that knockout of ASC gene could significantly inhibit the proliferation of PRV progeny virus.In this study,a PK15-ASC-/-cell line was successfully constructed,and in which the PRV infection was significantly inhibited.