Rho A-Rock 1信号通路对传染性脾肾坏死病毒感染的调控及作用

Regulation and effect of Rho A-Rock 1 signaling pathway on infectious spleen and kidney necrosis virus infection

【目的】探究传染性脾肾坏死病毒(ISKNV)复制增殖与Rho A-Rock 1通路的关系。【方法】采用实时荧光定量PCR(RT-qPCR)检测ISKNV感染CPB细胞12,24,48,72和96 h后病毒DNA拷贝数的变化;同时采用RT-qPCR和蛋白免疫印迹方法检测病毒感染CPB细胞12,24,48和72 h后Rho A与Rock 1转录及蛋白表达水平的变化;通过Rho A抑制剂(CCG-1423和Rhosin)、Rock 1抑制剂(Thiazovivin和Y-27632)及siRNA抑制Rho A-Rock 1通路,研究Rho A-Rock 1通路抑制对ISKNV复制增殖的影响。【结果】ISKNV感染12~48 h,病毒的DNA拷贝数无显著变化,感染72 h病毒DNA拷贝数显著上升,感染96 h病毒的拷贝数上升变缓,说明ISKNV感染72 h时病毒正处于复制增殖的高峰期。RT-qPCR及蛋白免疫印迹结果表明,ISKNV感染72 h时,Rho A和Rock 1 mRNA转录水平和蛋白水平均显著上调。Rho A抑制剂Rhosin和CCG-1423及Rock 1抑制剂Y-27632和Thiazovivin均可使ISKNV基因组拷贝数和病变的CPB细胞数显著下调;利用siRNA敲降Rho A和Rock 1时,ISKNV的基因组拷贝数也显著下调。【结论】Rho A-Rock 1信号通路可正调控ISKNV的感染,抑制Rho A-Rock 1通路可显著抑制ISKNV复制增殖。

【Objective】This study explored the relationship between infectious spleen and kidney necrosis virus(ISKNV)replication and Rho A-Rock 1 signaling pathway.【Method】RT-qPCR was used to detect the changes of viral DNA copy numbers at 12,24,48,72 and 96 h after infection of CPB cells with ISKNV.RT-qPCR and Western Blotting were used to detect changes of Rho A and Rock 1 transcription and protein expression in CPB cells at 12,24,48 and 72 h after virus infection.The effect of Rho A-Rock 1 pathway inhibition on ISKNV replication and proliferation was determined by Rho A inhibitors(CCG-1423 and Rhosin),Rock 1 inhibitors(Thiazovivin and Y-27632)and siRNA inhibition of Rho A-Rock 1 pathway.【Result】There was no significant change in DNA copy number of ISKNV from 12 to 48 h after infection,but it increased significantly at 72 h and slowed down at 96 h,indicating that peak of replication and proliferation was at 72 h.RT-qPCR and Western Blotting showed that the mRNA transcription levels and protein levels of Rho A and Rock 1 were significantly up-regulated at 72 h after ISKNV infection.Rho A inhibitors of Rhosin and CCG-1423 and Rock 1 inhibitors of Y-27632 and Thiazovivin significantly down-regulated copy number of ISKNV genome and number of cytopathic cells.Rho A and Rock 1 knockdown by siRNA also significantly down-regulated genomic copy number of ISKNV.【Conclusion】Rho A-Rock 1 signaling pathway positively regulated ISKNV infection and inhibition of Rho A-Rock 1 signaling pathway significantly inhibited ISKNV replication and proliferation.