羊口疮病毒B2L基因DNA疫苗的构建及其诱导的免疫应答  

Construction of DNA vaccine enconding B2L gene of Orf virus and its immune response induction

作　　者：李鹏飞[1] 冯将[1] 鲜思美[1,2] 刘嫒 李婷[1] 包细明[1] 张益 张素辉[3] LI Peng- fei;FENG Jiang;XIAN Si-mei;LIU Ai;IA Ting;BAO Xi-ming;ZHANG Vi;ZHANG Su-hui(College of Animal Science,Guizhou University ,Guiyang 550025,China;Institute of Animal Disease Research of Guizhou,Guiyang 550025,China;Academy of Animal Science of Chongqing , Rongchang, Chongqing 402460, China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025 [3]重庆市畜牧科学院,重庆荣昌402460

出　　处：《中国兽医学报》2018年第4期704-708,715,共6页Chinese Journal of Veterinary Science

基　　金：贵州省科学技术基金资助项目[黔科合LH字(2014)7667];贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队基金资助项目[黔科合人才团队(2015)4016];贵州省科技计划基金资助项目(黔科合支撑[2018]2264)

摘　　要：为研究所构建羊口疮病毒（OrfV）B2L基因DNA疫苗诱导小鼠的免疫应答效果，本研究对pMD18T-B2L质粒进行PCR扩增，克隆B2L片段至pVAX1载体中构建pVAX1-B2L重组质粒，进行酶切和测序鉴定；采用脂质体法将pVAX1-B21。真核表达质粒转染MDBK细胞，RTPCR和IFA法检测B2L基因在MDBK细胞中的转录和表达；将构建的DNA疫苗免疫KM系小鼠，采用间接ELISA、MTT和FACS法对其诱导的免疫应答进行研究。结果显示，成功构建pVAX1-B2L真核表达质粒，并在MDBK细胞中表达；免疫小鼠后，DNA疫苗能诱导小鼠产生Orfv特异性抗体；脾淋巴细胞增殖、CD4^＋、CD8^＋T淋巴细胞亚群百分比和IL-2、IFN-γ、IL-4细胞因子均高于pVAX1组和PBS组。结果表明，本研究制备的DNA疫苗能够诱导小鼠产生较高水平的体液免疫和细胞免疫应答。In order to construct DNA vaccine of Orf virus B2L gene and study its immune response induction in mice. The B2I. gene of pMD18T-B2L cloning plasmid was amplified by PCR and in serted into impressing vector pVAX1 to construction recombinant plamids pVAX1 B2L. The DNA vaccine plasmid was identified by PCR,digestion and sequencing. Then it was transferred into MD- BK cells by liposome 2000 and its transcription and expression in MDBK cells was detected by RT- PCR and IFA. KM mice were injected with DNA vaccine and its immune response induction was detected by indirect ELISA, MTT and FACS. The recombinant eukaryotic expression plasmids pVAX B2L was constructed successfully and could be expressed in MDBK cells. The special anti body of OrfV,proliferation of spleen lymphocytes,CD4^＋ ,CD8^＋ T cells,and the level of IL 2,IFN- γ and IL-4 in mice immunized with DNA vaccine were higher than pVAX1 empty vector and PBS control group. The DNA vaccine can induce a high level response of humoral and cellular immunity.

关 键 词：羊口疮病毒(OrfV) B2L基因 DNA疫苗 体液免疫 细胞免疫 

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