| Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most contagious animal virus diseases. It is one of infectious disease on the list of World Organization of Animal Disease (OIE) which must be reported. At present, seven different serotypes containing O, A, C, Asia1, and South African Territors1 (SAT1), SAT2 and SAT3 have been found and is prevalence in all over the world and it is also one of the most important infectious animal diseases in China. FMD serotype Asia1 and O had been spread in many part of China. Developed countries adopt the policy of culling in order to prevention and control the outbreaks of FMD, while developing countries adopt the policy of universal immunization. At present, inactivated vaccine was still widely used in our country. However, researchers found that there were serious security risks in inactivated vaccination. Therefore, the development of a safer, effective FMD vaccine has become a new focus on growing attention by researchers, included of recombinant subunit vaccines, multi-peptide vaccines, DNA vaccine and recombinant live vector vaccines.From the perspective of FMDV transmission, air-borne transmission have a great significance for its pandemic. Thereby it is important to increase the level of mucosal immune for prevention and control of FMDV. Live bacterial vaccines is one of wildly researched new type vaccines, and have the abilities to induced cellular immunity and humoral immunity. And live bacterial vaccines also have the abilities to stimulate mucosal immune responses. At present, there are many kinds carrier for the vaccines, including lactic acid bacteria, attenuation salmonella and so on. But there is little study on Bacillus subtillis and Saccharomyces cerevisiae which have the ability for expression exogenous gene and safety.In this research webservers, CTL Pred, Bce Pred and Harvard, have been used for predict the epitopes of FMDV structural protein VP1 and the high score epitopes have been choosen. At the same time the antigenic active site of FMDV serotype Asia 1 and O which have been reported was consulted. And then one team of multi-epitopes have been obtained and connected them by sequence of liner, GPGPG or AAA. So two box of multi-epitopes containing epitopes of FMDV serotype Asia1 and O, were designed and synthesized. Cholera toxin B subunit(CTB)and Th cell epitopes which come from FMDV (Th2) have been used as molecular adjuvant to construct multi-epitopes of FMDV serotype Asia 1 and O, CTEAs and CTEO. The secondary and tertiary structure were predicted through the software of molecular biology. The results show that there was little interference of neighboring epitopes in multi-epitopes of FMDV serotype Asia 1 and O and there are lots of epitopes located in protein surface. The antigenicity of multi-epitopes was higher and will become one of antigen for FMDV vaccine.Recombinant plasmids pSK-P1/2A-CTEAs, pSK-P1/2A-CTEO, pSK-CTEAs and pSK-CTEO containing capsid polypeptide P1-2A of FMDV and multi-epitopes of FMDV serotype Asia 1 and O have been constructed. Then based on the Pichia pastoris secretory expression vector pPIC9K, the recombinant plasmids pPIC9K-P1/2A-CTEAs, pPIC9K-P1/2A- CTEO, pPIC9K-CTEAs and pPIC9K-CTEO were constructed and expressed interest proteins multi-epitopes of FMDV serotype Asia 1 and O. Expression supernatant were purified by ammonium sulfate precipitation and evaluated the abilities for induce humoral and cellular responses in mice after intraperitoneal immunization. The result show that multi-epitopes were secreted into supernatant successfully and accounted for about 24%, 30%, 20% and 19% of the total supernatant protein. The results of immunization shows that multi-epitopes P1/2A-CTEAs, P1/2A-CTEO, CTEO and CTEAs have the abilities for stimulating specific serum antibody and inducing lymphocyte proliferation and secretory of IFN-γ. The results demonstrate that the subunit vaccines have the abilities of induce humoral and cellular responses, especially P1/2A-CTEAs (serotype Asia 1) and P1/2A-CTEO (serotype O).On this basis, the capsid polypeptide VP1-2A of FMDV serotype Asia 1 have been connected with multi-epitopes, and based on the Bacillus subtilis secretory expression vector pBC38C, which constructed by the work of predecessors, recombinant plasmids pBC38C-CTB- VP1/2A-CTEAs, pBC38C-CTEAs and pBC38C-CTEO were constructed. With electro- transformation recombinant plasmids were transformed into Bacillus subtilis 1A751, and live bacterial vaccines 1A751/CTB-VP1/2A-CTEAs, 1A751/CTEAs and 1A751/CTEO have been constructed. The expression of interest protein which have been secreted into supernatant accounted for about 21%, 17% and 15% of the total supernatant protein. The immune effectiveness of recombinant Bacillus subtilis were evaluated in mice after oral immunization (1010/each). Recombinant Bacillus subtilis 1A751/CTB-VP1/2A-CTEAs, 1A751/CTB-VP1/2A- Epi (constructed by predecessors), 1A751/CTEAs and 1A751/CTEO have the abilities to stimulate specific serum antibody and inducing lymphocyte proliferation and secretory of IFN-γ. At the same time the total mucosal antibody sIgA and specific mucosal antibody sIgA were significantly increased after oral immunization. The results demonstrated that oral immunization recombinant Bacillus subtilis not only have the abilities of induce humoral and cellular responses, but also have the abilities of induce mucosal immunity responses significantly, especially 1A751/CTB-VP1/ 2A-CTEAs (serotype Asia 1) and 1A751/CTB-VP1/2A- Epi (serotype O).As the widely use of antibiotics, the bacteria carriers such as Bacillus subtilis has been extremely limited. So in this research Saccharomyces cerevisiae which is not sensitive to antibiotics have been used for live bacteria vaccine. The recombinant plasmids (pYES2/CT-P1/ 2A-CTEAs, pYES2/CT-P1/2A-CTEO, pYES2/CT-CTEAs and pYES2/CT-CTEO) containing FMDV capsid protein P1-2A and multi-epitopes was constructed based on pYES2/CT, an expression vector of Saccharomyces cerevisiae and transformed into Saccharomyces cerevisiae INVSc 1. The target protein was expressed in the recombinant Saccharomyces cerevisiae (INVSc1/P1/2A-CTEAs, INVSc1/P1/2A-CTEO, INVSc1/CTEAs and INVSc1/CTEO), and the interest protein accounted for about 14%, 12%, 8% and 10% of the total soluble protein. The immune effectiveness of recombinant Saccharomyces cerevisiae were evaluated in mice after oral immunization (108/each). Recombinant Saccharomyces cerevisiae INVSc1/P1/2A- CTEAs, INVSc1/P1/2A-CTEO, INVSc1/CTEAs and INVSc1/CTEO have the abilities to stimulate specific serum antibody and inducing lymphocyte proliferation and secretory of IFN-γ. At the same time the total mucosal antibody sIgA and specific mucosal antibody sIgA were significantly increased after oral immunization. The results demonstrated that oral immunization of recombinant Saccharomyces cerevisiae not only have the abilities of induce humoral and cellular responses, but also have the abilities of induce mucosal immunity responses significantly, especially INVSc1/P1/ 2A-CTEAs (serotype Asia 1) and INVSc1/P1/2A-CTEO (serotype O).According to the immunization strategy of prime-boost, the effects of combined immunization were evaluated. The strategy of prime with DNA vaccine, pVAX-P1-2A-3C (serotype Asia 1) and pIRES3CP1 (serotype O), and boost with recombinant fowlpox virus, vUTAL-P1-2A-3C (serotype Asia 1) and vUTAL3CP1(serotype O)has been choosed, and oral live bacterial vaccines recombinant Bacillus subtilis 1A751/CTB-VP1/2A-CTEAs (serotype Asia 1)1A751/CTB-VP1/2A-Epi (serotype O) or recombinant Saccharomyces cerevisiae INVSc1/P1 /2A-CTEAs (serotype Asia 1) and INVSc1/ P1/2A-CTEO (serotype O) or mixture with both after each immunization. The result show that specific serum antibody, lymphocyte proliferation, IFN-γsecretory, total mucosal antibody sIgA and specific mucosal antibody sIgA of oral groups were significantly higher than the group of non-oral immunization groups. The results demonstrated that oral immunization live bacterial vaccines have the abilities to increase the effects of combined immunization which used immunization strategy of prime-boost, especially the mixture of recombinant Bacillus subtilis and recombinant Saccharomyces cerevisiae. |
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