猪O型FMDV重组多表位抗原基因的克隆表达及免疫学鉴定  

Cloning expression and immunological identification of recombination multi-epitope gene of FMDV type O in pig

作　　者：高明[1,2] 邵军军[2] 林彤[2] 赵付荣[2] 刘泽众 常惠芸[2] 吴润[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2017年第1期1-6,共6页Journal of Gansu Agricultural University

基　　金：“十二五”国家生猪现代产业技术体系项目(CARS-36-06B)

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.[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 BarnH I ,EcoR I and other sites, both genes were cloned into pPROEx-HTb vector to form a recombinant plasmid pE- IgG. 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 emul- sification of the mixture of proteins with a equal volume of several adjuvants including ISA206, ISA201, IMS1313,603, ISA61 to immunize BALB/e 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 multi- ple-epitope vaccine by emulsification of the mixture of proteins with ISA201 adjuvant to FMDV type O in pig was a promising vaccine candidate.

关 键 词：猪O型口蹄疫病毒 表位 佐剂 疫苗 

分 类 号：S852.43[农业科学—基础兽医学]