| Foot-and-mouth disease (FMD) caused by foot and mouth disease virus (FMDV) is a highly contagious and economically devastating disease, which always inflict cloven-hoofed animals such as cattle, sheep and pigs. There are 7 serotypes of FMDV, namely, O, A, C, SAT1, SAT2, SAT3 and Asia1. The type O is the most serious type, and spreads widely.Vaccination is the most effective way for the prevention of FMD and good vaccine is the precondition for the eradication of FMD. Inactivated vaccine maintains very good immunogenicity and plays important role in the prevention of the disease. But because of following risky factors such as the incomplete inactivation of the virus and escape of live virus, new type of vaccine which should be safer and more effective are under intensive study worldwide. With the development of molecular biology, more and more genetically engineering vaccines are being developed.Humoral immunity plays a crucial role in the inhibition and elimination of FMDV, although some other compound immunities may be primed during FMDV infection.Thus, the humoral immunity becomed not only the hinge when we designed the vaccine but also the sticking point that may be cast more important care in the incoming functional research. The major route of infection of FMDV includes pathways through the mucosa of alimentary system, respiratory system and skin. Therefore, the mucosal immune response against FMDV is considered in the design and development of FMDV vaccine.Cholera toxin (CT), which is the virulent factor from Vibrio cholerae, consists of one A subunit and five B subunits. The B subunit (CTB) is non-toxic to cells and attaches to cells by binding to ganglioside GM1. Presently, the CTB, one of most promising mucosal adjuvant, enhances organism immunity by the combination with antigen when delivered to mucosa.FMDV is a member of Aphthovirus genus of Picornaviridae family. The genome of FMDV is a positive RNA molecule of 8.5kb. The genome is composed of 5'UTR, 3'UTR and a large ORF between the UTRs. The large ORF contains L, P1, P2 and P3 gene. P1 gene encodes 4 major structure proteins of FMDV, namely, VP1, VP2, VP3 and VP4. VP1, VP2 and VP3 are the subunits of the capsid while VP4 locates in the interior of the viral particle. VP1 is the major antigen of FMDV and it contains three antigen sites of the B cell epitope, which is the major immuno-dominant epitope eliciting the protective humoral immunity. VP1 also contains some T cell epitopes, which are very important for the production of neutralizing antibody. And VP4, 3ABC and 3D protein contains epitopes that induce virus specific T-cell response.Base on the analysis of epitopes and immunity on FMDV, and the epidemiology of FMD, the fusion gene of CTB-Th2-VP1 and CTB-VP1-2A-Epi containing the Type O FMDV multi-epitopes gene, which composed of VP1 gene and other epitopes gene of FMDV, and the cholera toxin B subunit gene were constructed. The resulting fusion genes were cloned into pGEX-6P-1 expression vector. CTB-Th2-VP1 and CTB-VP1- 2A-Epi fusion protein were expressed in form of inclusion body in E. coli BL21, and accumulated to the level of 20.7% and 29.0% of total bacterial proteins, respectively. The results of western blot analysis also demonstrated that the fusion protein could be recognized by the anti-FMDV and anti-CT antibody, respectively. After inclusion bodies were denaturalized and refolded in vitro, the result of the fusion protein-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. The mice were inoculated by intraperitoneal injection with the two kinds fusion protein that were denaturalized and refolded in vitro. The levels of humoral, cellular and mucosal immune responses were detected. The proliferation ability of splenic lymphocyte stimulated by FMDV and ConA was detected, the result showed lymphocyte FMDV stimulating index in group CTB-Th2-VP1 and CTB-VP1-2A-Epi are higher than that in other group. The results of ELISPOT detection for number of IFN-γblot showed that the number of CTB-Th2-VP1and CTB-VP1-2A-Epi groups were higher than that of other groups. The titer of anti-FMDV IgG and IgA antibody in mice serum from group CTB-VP1- 2A-Epi were higher or same as that of inactivated vaccine group, and were higher than that of other groups. The sIgA level in gut and lung lavage fluid in two groups were just same as other groups. These results showed the fusion protein of CTB-VP1-2A -Epi was able to evoke strong humoral immune responses. Our work established a good foundation for further study on the new and effective FMDV vaccines.The CTB-VP1-2A-Epi gene was cloned into pBC38C, a expression vector of Bacillus subtilis. The recombinant plasmid pBC38C-CTB-VP1-2A-Epi was constructed and introduced into Bacillus subtilis 1A751 by electroporation. The BALB/c mice were inoculated by recombinant B. subtilis 1A751 strains contained pBC38C-CTB-VP1-2A-Epi and pBC38C-VP1-2A-Epi respectively. The levels of humoral, cellular and mucosal immune responses were detected. The proliferation ability of splenic lymphocyte stimulated by FMDV and Con A were detected. The results indicated that the stimulate index of two test groups were higher than that of control groups. The results of ELISPOT detection for number of IFN-γblot showed that the number of two test groups were higher than that of other groups. The IgG and IgA antibody against FMDV type O in immunized mice could be generated in group CTB-VP1-2A-Epi and VP1-2A-Epi. But the IgG and IgA antibody titers were lower than that of inactivated vaccine group(p<0.05).The sIgA level in gut and lung lavage fluid were higher than that of the group inoculated inactived vaccine.To evaluate the feasibility of combined immunization, the recombinant fowlpox virus vUTL3CP1, the DNA plasmid pIRES3CP1 and the commercial inactived vaccine of serotype O of FMDV were used to immunize the BALB/c mice in different combination with recombinant B. subtilis 1A751/pBC-CTB-VP1-2A-Epi. The results showed that the recombinant can enhance the immunity of the other vaccine. The best strategy of vaccine was DNA plasmid pIRES3CP1 inoculation first, and followed by boosting with the recombinant virus vUTL3CP1, and administration with recombinant B. subtilis. The results indicated that the high titers of antibody against FMDV serotype O can be detected, and also accompanied with high cellular and mucosal immune.These results showed that fusion to CTB increased the systemic, cellular and mucosal immune against FMDV, and provided the possibility of developing a cheaper and more efficient oral vaccine for FMD. |
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