Targeting Foot-and-mouth Disease Virus Structural Protein VP1 To DC-SIGN And Its Immunogenicity

Foot-and-mouth disease(FMD) is a highly contagious desease, which can infect cloven-hoofed animals and cause severe economic losses as well as political consequences. Dendritic cells(DCs), the most potent class of antigen present cells(APCs), play an important role in inducing the effective immune defense against FMDV. Currently, targeting DC to improve the the vaccinal immunogenicity is one of the important strategies in designing vaccines. In this study, the structural protein VPI of FMDV AF/72 strain was espressed and purified using eukaryotic baculovirus and E.coli prokaryotic expression system respectively. To prepare DC-SIGN targeted antigens, the VP1 protein was combined with le(x) oligosaccharide using heterogeneous bifunctional crosslinking reagent Sulfo-SMCC and biotinStreptavidin(SA) crosslinking system. And then, the immune effects of the DC-SIGN targeted VP1 were detected in mice. The results of this study as following:1. Expression and purification of FMDV structural protein VP1 using two kinds of expression system. First, the optimized and synthesized VP1 gene according to the preferences of insect cell codon was inserted into pFastBacTMHTB to construct the recombinant plasmid pFastBacTMHTB-VP1. Subsequently the recombinant plasmid p FastBacTMHTB-VP1 was transfected into Sf9 cells to obtain the recombinant baculovirus rBac-HTB-VP1. The results of Western blotting(WB) and indirect immunofluorescent assay(IFA) showed that VP1 protein was successfully expressed in Sf9 cells at low expression level. Second, the VP1 gene was cloned to E.coli expression vector pET-28 a to construct the recombinant expression plasmid pET28a-VP1, and then pET28a-VP1 was transformed into bacterium BL21 to induce the expression of the VP1 protein. The results of SDS-PAGE and WB indicated that VP1 protein can be expressed successfully in BL21, at much higher level than eukaryotic baculovirus expression system. Further, the prokaryotic expressed VP1 protein was purified successfully with good reactogenicity after process optimization.2. The preparation of DC-SIGN targeted antigen VP1-le(x). First, the VP1 protein was coupled to SA chemically using heterogeneous bifunctional crosslinking reagent Sulfo-SMCC, and the results of SDS-PAGE and WB showed that VP1 protein was successfully coupled to SA to form the SA-VP1 conjugates. Furthermore, SA in the conjugates was verified to be able to bind with bilotin specifically according to ELISA. Therefore, based on the characteristic of SA binding biotin with high affinity, we combined SA-VP1 conjugates with biotin modified by le(x) oligosaccharide, and then yielded DC-SIGN targeted antigen VP1-le(x).3. Evaluation of immune effect of VP1-le(x). VP1-le(x) was emulsified with Freund’s adjuvant and then was injected into mice to evaluate its immunogenicity. The purified VP1 protein, FMDV inactivated vaccines and PBS was used as negative, positive control and mock, respectively. Results showed that comparing with mock group, the other three groups were able to generate FMDV-specific antibodies, CD4 + and CD8 + T cells in peripheral blood rose a higher level to a certain degree, and T spleen lymphocyte proliferated significantly after antigen stimulation. Detection of cytokines chip showed that VP1-le(x) elicited much more IL-2, IFN-γ, IL-4 and IL-10 than positive and negative control group, while IL-2 and IFN-γ were more significant(p<0.01). Above results indicated that VP1-le(x) was able to elicit humoral immune response and cellular immune response simultaneously, but cellular immunity is higher. This study was to lay an experimental foundation for the development of novel targeted vaccine.