| 摘要: |
| 【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。 |
| 关键词: 脾肾坏死病毒 病毒复制 Rho A-Rock 1信号通路 |
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| 基金项目:广东省基础与应用基础研究基金项目(2021A1515012057)；广东省现代农业产业技术创新团队专项基金项目(2019KJ140, 2019KJ141)；广东省海洋经济发展（海洋六大产业）专项资金项目(GDOE［2022］A13) |
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| Regulation and effect of Rho A-Rock 1 signaling pathway on infectious spleen and kidney necrosis virus infection |
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TAN Youyan,NIU Yinjie,LI Ningqiu,et al
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| Abstract: |
| 【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 upregulated 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. |
| Key words: spleen and kidney necrosis virus viral replication Rho A-Rock 1 signaling |
