猪乙型脑炎病毒EⅢ与猪细小病毒VP2基因的串联表达及免疫原性  

Coexpression for E Ⅲgene of Japanese encephalitis virus and VP2 gene of porcine parvovirus and its immunogenicity

作　　者：李云云[1] 郭万柱[1] 徐志文[1] 林华[1] 李宇[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6期774-778,794,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：通过重叠PCR的方法将猪细小病毒(PPV)VP2基因和猪乙型脑炎病毒(JEV)EⅢ基因通过Linker连接,并克隆至真核表达载体pCI-neo,得到重组质粒pCI-VP2-EⅢ。将重组质粒pCI-VP2-EⅢ转染Vero细胞,其表达产物通过RT-PCR和间接免疫荧光试验的方法检测出目的基因的转录与表达。质粒pCI-VP2-EⅢ在无免疫佐剂参与的情况下,免疫6～8周龄的BALB/c小鼠,同时设空质粒接种组,猪细小病毒灭活苗和猪乙脑弱毒苗免疫组及磷酸盐缓冲液接种组作为参比对照。结果表明,pCI-VP2-EⅢ免疫组,在二免后1周产生JEV和PPV的特异性抗体,三免后达到高峰;对ConA有显著的反应性,且与对照组差异明显;通过流式细胞仪检测,CD3+T、CD4+T显著高于猪细小病毒灭活苗免疫组和对照组(P<0.05),略低于乙脑减毒苗免疫组,但CD8+T显著高于其他试验组(P<0.05),这表明pCI-VP2-EⅢ接种后可能主要是以CD8+T淋巴细胞介导的细胞免疫。E Ⅲgene of Japanese encephalitis virus and VP2 gene of porcine parvovirus were fused with linker by overlapping extension PCR and cloned into the eukaryotic expression vector pCI-neo.The obtained recombinant plasmid pCI-VP2-EⅢ was transformed into Vero cell and the expression product was identified by RT-PCR and indirect immunofluorscence assay.Mice were immunized with recombinant plasmid,in the absence of adjuvant,empty plasmid vaccinated group,inactivated vaccine against porcine parvovirus and swine attenuated Japanese encephalitis immunization group and phosphate buffered saline inoculated group were as the control reference.The result showed that recombinant plasmid of pCI-VP2-EⅢcould induce immunize reponse at 1w post-boosting immunization and the antibody levels reached a peak at post-three immunization;spleen lymphocyte proliferative response showed that recombinant bacteria on ConA reactivity significantly,and the significant differences between the control group;by flow cytometry,it was found the ratio of CD3＋ T,CD4＋ T in the mice of pCI-VP2-EⅢ group were significantly higher than porcine parvovirus and phosphate buffered saline inoculated group,with slightly lower than the attenuated Japanese encephalitis immunization group,CD8＋ T was significantly higher than other experimental group,suggesting that pCI-VP2-E Ⅲ after vaccination may be mainly induce CD8＋ T-lymphocyte mediated cellular immunity.

关 键 词：细小病毒 乙脑病毒 VP2基因 EⅢ基因 核酸疫苗 

分 类 号：S852.651[农业科学—基础兽医学]