Development Of Bivalent Vaccines Against Foot-and-mouth Disease

Foot-and-mouth disease (FMD), caused by a picoraavirus of the Aphtovirus genus (FMDV), is a highly contagious and economically devastating disease of swine, cattle and other cloven-hoofed animals. FMDV consists of different serotypes. Type O FMDV was the main that caused FMD in China, and type A often broke out. Research on bivalent vaccines against both type A and O FMDV is important. This research focused on the development of bivalent vaccine against FMDV using prokaryotic and yeast expression system, the development of bivalent DNA vaccine and improvement of the efficacy of FMDV DNA vaccine. The results are listed below:1. Development of bivalent vaccine against FMDV using E.coli expression system. According to sequence of both type A and O FMDV, the sequence of the epitopes of type A and O FMDV was determined. HBcAg cDNA was fused with FMDV epitopes to improve the immunogenicity. The fused gene was cloned into the prokaryotic expression vector pET28a. The right readframe was confirmed by DNA sequence. E. Coli BL21 (DE3) was transformed with the positive plasmid and induced to express the protein by IPTG A more protein band approximately 33 KD in the positive bacteria, which was in line with the expected molecular weight, than the transformed pET28a one was analyzed by SDS-PAGE. The protein was formed in inclusion body and its content was above 95 % after being purified with Ni+-NTA-Sepharose 6B. The purified protein was cofirmed its fidelity using MALDI-TOF Mass analysis. Specific antibody was proved to produce in the immunized mice. 2. Development of bivalent vaccine against FMDV using pichia pastoris expression system. The epitopes of type A and O, fused with HBcAg cDNA,were subcloned into pPIC9K, and transformed into GS115. The gene was proved to be intigated into ihepichia genome by Southern Blotting analysis. The positive clone, induced with 1% methanol, expressed the protein about 37 KD. Western blot analysis was employed for assaying its immunogenicity.3. Development of bivalent DNA vaccine against FMDV. The epitopes, fused with HBcAg cDNA, were cloned into the eukaryotic expression vector pcDNA3. Western blot analysis was employed for assaying the fused gene. Specific antibody and T cell proliferation was produced in the immunized mice.4. Genetic adjuvant of IL-1P 163-171 nanopeptide to improve the immunogenicity of FMDV DNA vaccine. A DNA fragment encoding thehuman IL-1 3 163-171 nanopeptide was fused to FMDV DNA vaccine, andinjected into mice to analyze its immune responses. Compared with the control mice which received DNA vaccine alone, significant increase in not only the specific antibody response (about 4 fold for both A and O FMDV)but also in T cell proliferation(about 2 fold after the A or O type FMDV stimulation) wasobserved in mice which received IL-1 3 163-171-FMDV DNA vaccine.5. Genetic adjuvant of IL-2 to improve the immunogenicity of DNA vaccine. IL-2 gene, fused to FMDV DNA vaccine, caused eightfold increase both O and A FMDV antibody, and twofold increase T cell proliferation. When the IL-2 genetic adjuvant together with the DNA vaccine injected at the same place caused fourfold increase both O and A FMDV antibody, and twofold increase T cell proliferation. But if the IL-2 genetic adjuvant and DNA vaccine were injected at the different place respectively, the genetic adjuvant will notincrease the efficacy of DNA vaccine.6. Genetic adjuvant of IL-4 to improve the immunogenicity of DNA vaccine. IL-4 cannot be used as genetic adjuvant to improve the FMDV specific-antibody produce. IL-4, fused with DNA vaccine, increase T cell proliferation about twofold; while threefold when IL-4 and DNA vaccine were injected at the same place. But if the IL-4 genetic adjuvant and DNA vaccine were injected at the different place responctively, IL-4 would not increase T cell proliferation.7. Coimmunization of Agaricus blazei Murill extract to improve the immunogenicity of DNA vaccine. Agaricus blazei Murill extract caused about fourfold increase the specific antibody, a...