Ubiquitination Of Recombinant VP1-VP4Protein Of FMDV In Dendritic Cells

Foot-and-mouth disease (FMD) is a highly contagious disease affectingcloven-hoofed animals. FMDV have seven serotypes(A, C, O, SAT1, SAT2, SAT3andAsia I) with a large number of subtypes, there is not possess cross protection among sevenserotypes.FMDV capsid consisting of VP1, VP2, VP3and VP4has icosahedral structure. TheVP1, VP2and VP3are the major FMDV interface elements, major antigenic sites presenton the VP1section GH loop at amino acid140to160enriched B cell epitope, are the fourstructural proteins stimulate the body humoral immune response to the main ingredient, butthe main area is also mutated VP1and only use this one as an antigen epitope generated bystimulating the immune protection of animals very short duration. In addition VP1is alsorich in T cell epitopes from type O FMDV VP1stimulation of CTL responses generatedcattle rest six kinds of attacks FMDV has a protective effect, suggesting that cross-protective immunity can play T-cells containing VP1epitope. Although located inside thecapsid VP4, but there are T, B-cell epitopes, and conservative amino acid sequences, avariety of MHC haplotype can identify VP4of20-34amino acid sequence, it isconsidered to be the preparation of common VP4one preferred type of FMD vaccineantigen. Based on this, we designed the FMDV VP1-VP4antigen fusion protein is desiredto obtain not only to stimulate the body to produce a more comprehensive and effectiveimmune response (including humoral and cellular immune response) and the differentserotypes of the virus can exhibit cross-protective immunity effect.Protein ubiquitination involve in many important physiological functions and iswidespread in cell. One of the earliest determined function is the ubiquitin-mediatedprotein degradation. There have two major protein degradation pathways: lysosomepathway and the proteasome pathway. Different types of Ubiquitination determines the fateof the substrate proteins. Previous studies suggest that poly-ubiquitination (K11, K48)protein tend to be transported to the proteasome for degradation, single-chain proteinubiquitination tend to be degraded in lysosome. Dendritic cells exist twoantigen-presenting ways: MHC-Ⅰ class pathway, MHC-Ⅱ class pathway in cells withantigen ubiquitination is closely related to the type of research FMDV antigen in the celltype used ubiquitination to reflect its transporter in the cells, the degradation pathway is aneffective method.In this study, FK1, FK2two antibodies detected dendritic cell lysates FMDVVP1-VP4fusion protein ubiquitination type. Test results showed that FMDV VP1-VP4fusion protein in DCs simultaneously sheets chain poly-ubiquitination, but proteins are poly-ubiquitination was significantly higher than the level of the single-strandedubiquitination. To further explore the possible reasons for this result caused, we usemannose MR competitive inhibition activity of the laser and confocal fluorescencemicroscopy dynamic changes in the antigenic cells. The results showed that the inhibitionof MR, BMDCs intake VP1-VP4fusion protein is not only not reduced but increased,suggesting MR in BMDCs VP1-VP4fusion protein intake process plays a negativeregulatory role. In order to verify the effects of ubiquitination on antigen presentationpathway inhibitors were blocking experiment using MHC antigen presentation of MHC-Imolecules and antigen presentation pathway-Ⅱmolecular pathway. When the MHC-Imolecules inhibit the pathway discovery and without inhibitor group compared to thenormal number of15min and30min time point during the two endocytic cell bodiesincreased significantly, into lysosomes by endocytosis bodies of VP1-VP4protein alsoincreased significantly, time was extended to60min after even a great form of lysosomalvesicles. Inhibitors block the antigen through the use of proteasome degradation pathway,lysosomal degradation caused by the amount of antigen was significantly increased. Theresults demonstrate that FMDV VP1-VP4fusion protein through ubiquitination-proteasome pathway is degraded cells, BMDCs can be cross-presentation pathway ofantigen presenting FMDV VP1-VP4. When suppress MHC-Ⅱmolecular pathway foundthat due to the presence of inhibitors of lysosomal function is inhibited, BMDCs fusionprotein intake can not be effectively degraded in lysosomes, with the extension of timewithin the cell endosome/dissolved an increase in the number of body enzymescontinuous integration, form larger, VP1-VP4protein in the cell with time and stranded.It can be proved, FMDV VP1-VP4fusion proteins can be degraded in lysosomes,MHC-II molecules proposed pathway is important BMDCs proposed FMDV VP1-VP4antigen.In this study, we demonstrated that FMDV recombinant proteins VP1-VP4can besingle and poly ubiquitinated in BMDCs and degraded in lysosomal and proteasomepathway. we use of western blot, confocal fluorescence microscopy imaging techniques toshow up the pathway of BMDCs processing、presenting FMDV VP1-VP4. These resultsfurther illustrate the body’s immune response against FMDV molecular mechanisms toprovide theoretical guidance for vaccine design, new technologies may provide support forthe comprehensive control of foot and mouth disease, and promote the healthy and rapiddevelopment of China’s animal husbandry.