Construction of Porcine Derived Rab Gene shRNA Libraries and Stable Cell Lines and the Effects on PRV Proliferation
[Objective]The purpose of this study was to investigate the role of Rab family proteins in the infection of Porcine pseudorabies virus(PRV)in host cells.[Method]Porcine renal epithelial cells(PK15)were used as the test model to construct Rab gene shRNA library,and purinomycin was used to screen stable cell lines that inhibited porcine Rab gene expression.Real-time quantitative PCR was used to detect cell knockdown efficiency,and flow cytometry was used to detect the effect of knockdown Rab gene on PRV proliferation.Western blotting was used to detect the expression levels of PRV-gB protein,and Real-time quantitative PCR was used to detect the expression levels of PRV-gB,PRV-TK genes,and related inflammatory factors after Rab gene knockout.[Result]A shRNA library with 13 Rab gene knockdown cell lines was successfully constructed.Flow cytometry analysis showed that knockdown of Rab gene significantly inhibited PRV-GFP proliferation(P＜0.05).Viral titer and Western blotting test results showed that Rab gene knockdown extremely significantly inhibited the generation of progeny virus and the expression of PRV-gB protein(P＜0.01).The results of Real-time quantitative PCR showed that the expression of PRV-gB and PRV-TK genes could be significantly inhibited after knocking down Rab gene(P＜0.05 or P＜0.01).The expressions of IFN-β,ISG15,IL-1β and IL-18 genes were significantly or extremely significantly increased after knocking down Rab14 and Rab27 genes(P＜0.05 or P＜0.01).[Conclusion]Knockdown of Rab gene could inhibit PRV proliferation.The results laid a foundation for further research on the role of Rab gene in PRV life cycle.
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