【摘要】 【目的】制备猪O型FMDV广谱多表位疫苗,并筛选最适合的免疫佐剂.【方法】选取O型FMDV 3个毒株VP1蛋白的优势表位,设计并合成重复串联表位基因3FoEN2,并克隆猪IgG重链恒定区基因.利用BamHⅠ,EcoRⅠ等位点将2个基因依次克隆到pProEX-HTb载体,构建重组质粒pE-IgG并转化大肠杆菌BL21(DE3)感受态细胞.以IPTG诱导表达得到融合蛋白pE-IgG,经SDS-PAGE电泳分析,Western-blotting鉴定.分别用5种佐剂ISA206、ISA201、IMS1313、603、ISA61乳化融合蛋白配制疫苗免疫BALB/c雌鼠,间接ELISA方法测定抗体水平.【结果】重组蛋白以包涵体形式正确表达,大小为45kU,且能与感染O型FMDV的猪的阳性血清发生特异性免疫反应;ISA201佐剂试验组刺激机体产生的抗体水平最高.【结论】pE-IgG蛋白具有很强的免疫原性,与ISA201佐剂混合制备成的猪O型口蹄疫病毒多表位疫苗可刺激动物机体产生高水平抗体,是具有良好开发前景的疫苗.更多还原

【Abstract】 【Objective】To prepare the broad spectrum multiple-epitope vaccines to FMDV type O and select the most suitable immune adjuvant.【Method】Selecting dominant epitopes of VP1 protein from three representative strains of FMDV type O to design and chemically synthesize a tandem repeat DNA fragment named 3FoEN2 and clone the gene of heavy chain constant region of pig IgG.By using the BamH Ⅰ,EcoRⅠ and other sites,both genes were cloned into pPROEx-HTb vector to form a recombinant plasmid pEIgG.A chimeric protein named pE-IgG was obtained by transforming the pE-IgG into Escherichia coli BL21(DE3)host cell and expressing induced by IPTG.,which was identified by SDS-PAGE electrophoresis and Western blotting.Five different multiple-epitope vaccines to FMDV type O in pig were developed by emulsification of the mixture of proteins with a equal volume of several adjuvants including ISA206,ISA201,IMS1313,603,ISA61 to immunize BALB/c female mice,and measure antibody level by indirect ELISA.【Result】The recombinant proteins were expressed as a formation of inclusion bodies in E.coli and the size of recombinant expression vector pE-IgG was 45 kU.Western blotting results showed that the recombinant pE-IgG was able to react with anti-FMDV positive serum.The highest antibody level was produced by ISA201 adjuvant trial group.【Conclusion】The immunogenicity of pE-IgG protein was strong.The multiple-epitope vaccine by emulsification of the mixture of proteins with ISA201 adjuvant to FMDV type O in pig was a promising vaccine candidate.更多还原