【作者】 胡博；

【导师】 金宁一；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 本研究利用网络服务器对多株Asia 1型、O型FMDV结构蛋白VP1进行表位预测,同时参考已知的Asia 1型、O型FMDV抗原活性位点,设计并合成FMDV多表位盒。将分子佐剂CTB和FMDV辅助性T细胞表位连入多表位盒中,构建Asia 1型、O型FMDV复合多表位基因盒CTEAs和CTEO。将Asia 1型、O型FMDV衣壳蛋白前体P1/2A分别与复合多表位基因相连,利用毕赤酵母进行分泌表达,将纯化的表达产物P1/2A-CTEAs、P1/2A-CTEO、CTEO和CTEAs腹腔免疫小鼠,证明复合多表位均可刺激小鼠高水平的体液免疫和细胞免疫的产生,说明设计的FMDV复合多表位具有良好的免疫原性,其中尤以P1/2A-CTEAs(Asia 1型)和P1/2A-CTEO(O型)免疫组效果最佳。将Asia 1型FMDV复合多表位基因与其结构蛋白VP1-2A基因相连,利用枯草芽孢杆菌分泌型表达载体pBC38C,构建了可稳定表达目的蛋白的重组枯草芽孢杆菌1A751/CTB-VP1/2A-CTEAs、1A751/CTEAs和1A751/CTEO,并以口服的方式(1010/只)进行小鼠免疫实验,并检测特异性血清抗体、T淋巴细胞的增殖和IFN-γ的分泌、黏膜总sIgA和黏膜特异性sIgA。证实重组枯草芽孢杆菌既可刺激体液免疫和细胞免疫的产生,同时对机体黏膜免疫也有显著地诱导作用,其中以重组枯草芽孢杆菌1A751/CTB-VP1/2A-CTEAs口服免疫效果最佳。构建了稳定表达FMDV复合多表位的重组酿酒酵母INVSc1/P1/2A-CTEAs、INVSc1/P1/2A-CTEO、INVSc1/CTEAs和INVSc1/CTEO,并以口服的方式(108/只)进行小鼠免疫实验。说明重组酿酒酵母不仅可以刺激体液免疫和细胞免疫的产生,而且对机体黏膜免疫也有显著地诱导作用,其中以重组酿酒酵母INVSc1/P1/2A-CTEAs和INVSc1/ P1/2A-CTEO口服免疫效果最佳。利用prime-boost免疫策略对多种疫苗联合免疫效果进行研究。以本室构建的Asia 1型、O型FMDV核酸苗pVAX-P1-2A-3C(Asia 1型)和pIRES3CP1(O型)为首免,使用重组鸡痘病毒vUTAL-P1-2A-3C(Asia 1型)和vUTAL3CP1(O型)加强免疫,并于每次免疫的同时灌服本实验中效果较好的重组活菌载体疫苗。明确口服活菌载体疫苗能显著提高prime-boost免疫策略下疫苗联合免疫的效果,显著提高机体细胞免疫、体液免疫以及黏膜免疫水平,其中以重组枯草芽孢杆菌和重组酿酒酵母混合制剂口服免疫效果最佳。更多还原

【Abstract】 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.更多还原