T Lymphocyte Response Triggered By Dendritic Cells Pulsed With Protein Antigen Of FMDV VP4

Foot-and-mouth disease(FMD) is caused by FMDV virus(FMDV), whichi is an acute, febrile, highly contagious vesicular disease of cloven-hoofed animals. The antigen of FMDV is so easy to variate,so that there is no effective preventive measures. VP4 do not variate in the fore kinds of FMDV structural proteins. And it is proved that FMDV structural protein 1A(VP4) is rich in antigetic molecules of T cells and B cells antigetic epitopes. However, the antigen-presenting mechanism for FMDV VP4 are still unclear. Dendritic cells(DCs) are the most powerful professional antigen-presenting cells in the body at present.DCs were not largely susceptible to infection by integrin-binding FMDV but were susceptible to culture-adapted virus. Binding specific antibodies to integrin-binding FMDV at neutralizing or subneutralizing IgG concentrations significantly enhanced infection via CD32 (FcγR). Infection of moDC by the FMDV IC was productive and associated with high levels of cell death. Infected moDC were unable to efficiently stimulate FMDV-specific CD4+ memory T cells, but exposing moDC to IC containing inactivated FMDV resulted in significantly increased T cell stimulation. Activated T cells can differentiate into Th1 subset with the stimulation of IL-12. Th1 cells are able to mediate cellular immune response and promote opsonised-antibodies(IgG), which clear the infection of intracellular pathogens. In the role of IL-4, Th cells is conducive to Th2 cell differentiation, which mediate humoral immune response to produce neutralizing antibodies to clear the pathogen in the peripheral circulation. Currently, how dendritic cells initiate T cell response to FMDV remains elusive.In this study, we accord〝the experimental activities of animal pathogens required for biosafety rules〞published by Ministry of Agriculture in december 12, 2008, neither use live virus, nor use inactivated virus.Firstly, we synthesize pBluescriptⅡSK(+)-VP4 according the published foot-and-mouth disease virus nucleotide sequence in GenBank, then pBluescriptⅡSK(+)-VP4 were transfered into the pET-32a (+) prokaryotic expression vectors through BamHI and Xho I sites and construct the prokaryotic expression vectors of pET-32a-VP4.Recombinant expression vectors of VP4 were transformed into host bacteria BL21 for preparation of engineering bacteria. Optimize the expression conditions and the expression products were detected by SDS-PAGE to determine the optimal protein expression condition and to do Western blot via His-Tag of the vector. Purified VP4 protein antigens through electric elution. The immature DCs which were pulsed with FMDV VP4 protein antigens cocultured with T cells, and the DCs + T and the DCs as two controls. T cells were stimulated for 3h, 9h, 24h and 48h and culture supernatants from in vitro FMDV VP4-stimulated T lymphocytes were assayed for IFN-γand IL-4 contents, in order to elucidate the activitation effects of FMDV VP4 antigentic protein to T cells.The experimental results showed that the pET-32a-VP4 gene was correct by identification with Xho I and NcoⅠdigestion, and was consistent with the Gene Bank published sequence Foot-and-mouth disease virus O isolate O/NYOO and the homology was 100%. The proteins that express by host bacterium BL21 were detected SDS-PAGE, which indicated we can got the largest amount, when the IPTG concentration of 1mmol/L, 5 h after induction. We can see 25kDa bands after Western blotting, consistent with the expected size of the target protein.The ELISA results for cell co-culture showed that the DC pulsed with FMDV VP4 protein activated the T cells efficiently. These results not only lay a foundation for further study the antigen-presenting pathways in DCs for FMDV VP4 protein antigen, but also provide theoretical guidance for the design and development new vaccines.