携带猪乙型脑炎DNA疫苗减毒沙门菌的构建及其免疫原性  

Construction and immunogenicity of attenuated Salmonella choleraesuis C500 harbouring Japanese encephalitis virus DNA vaccine

作　　者：李云云[1] 郭万柱[1] 徐志文[1] 韩国全[1] 林华[1] 漆信桥[1] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安625014

出　　处：《中国兽医学报》2011年第5期697-702,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30500019);四川省教育厅重点实验室基金资助项目(07ZZ027);"长江学者和创新团队发展计划"创新团队资助项目(IRT0848)

摘　　要：将乙型脑炎病毒E蛋白主要抗原片段基因串联,构建真核表达载体pCI-EAB。将重组质粒pCI-EAB转染Vero细胞,表达产物通过RT-PCR和间接免疫荧光试验可检测出目的基因的转录与表达。质粒pCI-EAB电转化入减毒猪霍乱沙门菌,观察重组菌的安全性、体内外的稳定性与免疫原性。动物试验表明,重组菌是相对安全和稳定的;重组菌S.C500/pCI-EAB体外培养时,D600值在0.8左右质粒是比较稳定的;以1×108CFU剂量的重组菌S.C500/pCI-EAB口服免疫小鼠,在二免后1周和三免后1周,重组菌抗体水平与S.C500/pCI-neo空载体组差异显著(P<0.01);脾脏淋巴细胞增殖情况显示,重组菌对ConA有显著的反应性,且与对照组差异明显,同时CD3+、CD4+和CD8+T细胞的数量也显著高于对照组(P<0.01)。上述结果初步表明,以减毒沙门菌为载体的猪乙型脑炎DNA疫苗具有良好的安全性、稳定性和免疫原性,为猪乙型脑炎口服活菌疫苗的研制奠定了基础。In tandem with major antigenic segment of E gene were cloned into eukaryotic expression vector pCI-neo,then the recombinant plasmid named pCI-EAB was obtained.The recombinant plasmid pCI-EAB was transformed into Vero cell and the expression of recombinant plasmid was identified by RT-PCR and indirect immunofluorscence assay.Then pCI-EAB was electroporated into the attenuated Salmonella choleraesuis C500 and designated as S.C500/ pCI-EAB.Its security,stability and immunogenicity were observed.The animal experiments showed that the recombinant bacterium was relatively safe and stable;the recombinant bacterium S.C500/pCI-EAB was cultured in vitro,D600 nm around 0.8 plasmid was relatively stable;BALB/c mice were immunized orally with S.C500/pCI-EAB harboring recombinant plasmid at the dosage of 108 and boosted one week later with the same dose,for a total of three times.The S.C500/pCI-EAB could induce significant humoral immune response in mice compared with the control（P0.01） at 1 weck post-boosting and 1 weck post-three immunization;spleen lymphocyte proliferative response showed that the recombinant bacterium reacted to ConA,and the significant differences with the control group;flow cytometry displayed that CD3＋T,CD4＋T,CD8＋ T ratio in the mice of S.C500/pCI-EAB group were significantly higher than the control group（P0.01）.The results indicated that the attenuated Salmonella choleraesuis C500 as an oral delivery vector for DNA vaccine is of safety and stability and immunogenicity.S.C500/pCI-EAB vaccine strain could probably serve as a vaccine against Japanese encephalitis virus.

关 键 词：猪乙型脑炎病毒 E蛋白主要抗原片段 减毒沙门菌 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]