| ObjectiveIn this study,the models of the RNA interference system for green fluorescent protein and dual luciferase genes were established to screen the RNAi inhibitors of Chinese sacbrood virus(CSBV).In order to explore the mechanism of CSBV interference inhibitor,the gel migration,GST pull-down and Far-western blotting experiments were implemented,and this study provide new ideas to research CSBV to escape RNAi of Apis cerana.MethodspshRNA-GFP and GFP-si RNA were co-transfected with GFP into 293 T cells,then the fluorescence intensity and protein expression level of GFP protein in 293 T cells were detected by fluorescence microscope and Western blot,respectively.At the same time,the recombinant plasmid p SG5 m P19 of the tomato bushy stunt virus protein P19 was also co-transfected with GFP into293 T,as a positive control.On the based of this RNAi model,the recombinant plasmids p TT5-VP1,p TT5-VP2,p TT5-VP3 and p EGFP-N1 were co-transfected with pshRNA-GFP or GFP-si RNA into 293 T cells to screen the CSBV structural protein VPX which can inhibit RNAi,respectively.In order to quantitatively analyze the inhibition efficiency of protein VPX,pshRNA-Luc and si RNA-Luc of dual luciferase(Luc)were cotransfected into 293 T cells with p GL3 and p RL-TK respectively to construct dual luciferase RNAi system.Through this model,p TT5-VPX,PGL3,and p RL-TK was co-transfected with pshRNA-Luc and si RNA-Luc in 293 T respectively to test the inhibitory efficiency of VPX.Based on the above results,in order to explore the mechanism of VPX inhibit RNAi,Dicer enzyme and p GL3 double stranded RNA(ds RNA-p GL3)with different lengths were incubated with VPX protein,then the effect of VPX protein on the cleavage function of Dicer enzyme was detected by gel migration test(EMSA).At the same time,the expression plasmid p GEX-6p-1-Ago2 was constructed and the recombinant protein Ago2 was purified,then GST pull-down and Far-western blotting were applied to verify the interaction between r Ago2 and VPX proteins.ResultsThe fluorescence intensity and expression level of GFP protein in 293 T cells were significantly decreased by fluorescence microscope and Western blot,and the green fluorescence intensity was restored after adding P19 protein,which indicated that RNAi model was successfully constructed.Then,to screen the inhibitory effects of CSBV structural proteins VP1,VP2 and VP3 by this system,the results showed that structural protein VP3 can significantly restore the green fluorescence intensity by sh RNA and the expression level of GFP protein,indicating that CSBV VP3 protein has the inhibition function to RNAi.In order to quantitatively analyze the inhibitory efficiency of VP3,pshRNA-Luc plasmid and three synthesized si RNA1,si RNA2 and si RNA3 of Luc were added to the constructed Luc expression RNAi system and all can inhibit the activity of dual luciferase.Among them,when the ratio of pshRNA-Luc and PGL3(p RL-TK)is 0.5:1,the inhibition rate can reach 60%.Among the three Luc si RNAs,the inhibition rate of si RNA3 was the highest which could reach 79%.After adding the p TT5-VP3 plasmid,the activity of dual luciferase inhibited by sh RNA was restored to 149%,while the activity of dual luciferase inhibited by si RNA3 had no significant recovery.In order to further study the possible mechanism of VP3’s inhibition function of RNAi,GST pull-down and Far-western blotting experiments were performed on p GEX-6p-1-Ago2 and p ET-28a-VP3.The EMSA experiment was performed on Dicer enzyme and recombinant protein VP3 after incubation.The results showed that VP3 can inhibit the activity of Dicer enzyme and exert an inhibitory effect on RNAi.Conclusions1.The models of GFP and dual luciferase gene RNAi systems were applied to study the RNAi inhibition effect of CSBV structural protein,and the results show that CSBV structural protein VP3 has RNAi inhibition function.2.CSBV structural protein VP3 mainly could reduced Dicer enzyme activity of cleaving ds RNA,and inhibit RNAi function of Apis cerana. |
PDF Full Text Request