The Study Of The Interaction Between The VP0Protein And Pocine IFITM3Protein

Foot-and-mouth disease (Foot and Mouth Disease, FMD) is a zoonotic acute, febrile, highly contagious viral infection of cloven-hoofed animals, it mainly causes the fever and the formation of blisters and ulceration on limbs, mouth, tongue, nose, breasts and other parts. Foot-and-mouth disease virus (Foot and Mouth Disease Virus, FMDV) genome is a single-stranded positive-strand RNA encoding12mature viral proteins, in which the VPO protein are one of the structural proteins of the virus, and the VPO protein is closely related to the virus entry.Our laboratory’s study before have found that sIFITM3protein has antiviral activity. In order to find the mechanism the sIFITM3protein inhibits the FMDV replication, this study verifies the interaction between sIFITM3and VPO protein by applying Yeast Two-Hybrid, Cell colocalization technique and GST-Pull Down technique. To analyze the domains that sIFITM3interacts with VPO, sIFITM3cDAN was divided into different segemts. The sIFITM3was expressed in fragments in293T cells, and the sIFITM3segements that interact with VPO was identified with co-immunoprecipitation. The sIFITM3was expressed in fragments in BHK cells, and the antiviral activity was identified with plaque assay.To acquire the VP2and sIFITM3protein, our study constructs the rBacmid-VP2, rBacmid-FVP2, rBacmid-PsIFIMT3, rBacmid-sIFITM3(1-165), rBacmid-FsIFITM3(1-165).1. Analyzing the interaction between FMDV VPO and sIFITM3proteinAmplification of the gene FMDV VPO and sIFITM3amino terminal extracellular outer zone nsIFITM3by PCR, digested with EcoR I and Sal I and ligated into the yeast expression vector pGBKT7and pGADT7and obtaining the recombinant plasmid pGBKT7-VP0, pGBKT7-nsIFITM3, pGADT7-VP0and pGADT7-nsIFITM3.The plasmids were digested by EcoR I, Sal I and EcoR I, XhoI. The results show that the recombinant plasmids are correct. The pGBKT7-VPO, pGADT7-nsIFITM3and the pGBKT7-nsIFITM3, pGADT7-VPO plasimids were transformated to yeast Y2HGold, the results show that VPO interacts with nsIFITM3.To identify the interaction between the VPO and sIFITM3protein, recombinant plasmids pFlag-VPO and pLenti7.3-sIFITM3-V5were co-transfected into293T cells. By indirect immunofluorescence assay after48h, observing in fluorescence confocal micro scope showed that VPO protein were widely distributed in the cytoplasm and membrane and the sIFITM3protein were distributed on the cell membrane. VPO and sIFITM3protein were in the same position of the cell.For overexpression of sIFITM3and VPO proteins, prokaryotic expression systems is used.Purification of inclusion body, obtaining the recombinant proteins. The GST-Pull Down experiments showed that sIFITM3protein did not interact with VPO protein. Analysing the reason:probably that purificated inclusion protein changed its protein strcture.To verify sIFITM3domain that interacts with VPO protein, our study construts a series of partial deletion sIFITM3expression plasmids, pLent7.3-sIFITM3(31-432)-V5, PLenti7.3-IFITM3(61-432)-V5, pLenti7.3-IFITM3(91-432)-V5, pLenti7.3-IFITM3(121-432)-V5, pLenti7.3-IFITM3(151-432)-V5, and pLenti7.3-IFITM3(1-165)-V5. The deletion expression plasmids and pFlag-VPO plasmid were co-transfected to293T cells, co-immuneoprecipitation experiments found that sIFITM3protein which were not deleted affected the interaxtion with VPO protein.2. The antiviral activity of different truncated sIFITM3protein expressed in the BHK cellsAs different truncated sIFITM3proteins influence the interaction with VPO protein, transfecting pLent7.3-sIFITM3-V5, pLent7.3-sIFITM3(31-432)-V5, pLenti7.3-IFITM3(61-432)-V5, pLenti7.3-IFITM3(91-432)-V5, pLenti7.3-IFITM3(121-432)-V5, pLenti7.3-IFITM3(151-432)-V5, and pLenti7.3-eGFP-V5to BHK cells, the plaque assay show that only the whole sIFITM3protein have obvious activity to FMDV infection.3. The expression of sIFITM3and FMDV VP2protein in Baculovirus expression systemVP2, FVP2, PsIFITM3, sIFITM3(1-165) and FsIFITM3(1-165) segments were cloned into pFBD pH-HA-NA-M1vector by digesting with Hind Ⅲ and Xho Ⅰ to obtain recombinant transfer plasmids pFBD-VP2, pFBD-FVP2, pFBD-PsIFITM3pFBD-sIFITM3(1-165), pFBD-FsIFITM3(1-165).The transfer vectors were transformed into DH1OBac competent cells, extracted bacmids after the blue-white screening and purification, transfected bacmids into Sf9cells to obtain recombinant baculovirus rBacmid-VP2, FVP2, PsIFITM3, sIFITM3(1-165), FsIFITM3(1-165). The recombinant baculovirus infected the Sf9cell, our study analyzed the protein expression of the baculovirus. This study was the foundation of the SPR assay and the crystallization assay.