表达猪附红细胞体ENO基因的质粒DNA的构建及免疫效果研究  

Construction and Immunological Effect Analysis of Plasmid DNA Expressing ENO Gene of Mycoplasma suis

作　　者：赵云 伍生军[1] 倪婷婷[1] 王淼 张守发[1] 许应天[1] 薛书江[1] ZHAO Yun;WU Shengjun;NI Tingting;WANG Miao;ZHANG Shoufa;XU Yingtia;XUE Shujiang(Agricultural College of Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学农学院,延吉133002

出　　处：《中国畜牧兽医》2018年第9期2559-2565,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31460657);吉林省科技厅青年科研基金(20150520129JH)

摘　　要：试验旨在构建表达猪附红细胞体ENO基因的质粒DNA,并测定其免疫效果。将猪附红细胞体ENO基因克隆到PVAX1真核表达载体上,然后转染到Vero细胞中进行表达并测定其免疫效果。将18只BALB/c小鼠(雌雄各半)随机分为3组(PVAX1-ENO质粒DNA免疫组、PVAX1空载体对照组及PBS对照组),每组6只,每组小鼠对应免疫接种PVAX1-ENO质粒DNA、PVAX1空质粒和PBS。分离血清并通过ELISA方法测定血清中ENO蛋白抗体效价、IgG1和IgG2a抗体水平及IFN-γ和IL-4细胞因子水平。三免2周后每组选取3只小鼠检测CD4+和CD8+含量。结果显示,本试验成功构建了PVAX1-ENO质粒DNA,经PCR和酶切鉴定正确,并能在Vero细胞中成功表达。PVAX1-ENO质粒DNA免疫组ENO蛋白抗体水平、IgG1和IgG2a抗体水平、IFN-γ和IL-4细胞因子水平及CD4+和CD8+含量均显著高于PVAX1空载体对照组及PBS对照组(P<0.05)。结果表明,PVAX1-ENO质粒DNA可显著提高BALB/c小鼠的体液免疫水平,并在一定程度上刺激BALB/c小鼠细胞免疫。The aim of this experiment was to construct the plasmid DNA expressing ENO gene of Mycoplasma suis and determine its immune effect.The Mycoplasma suis ENO gene was cloned into PVAX1 eukaryotic expression vector,then transfected into Vero cells to express and determine its immune effect.18 BALB/c mice(half male and female)were randomly divided into 3 groups(PVAX1-ENO plasmid DNA immunization group,PVAX1 empty vector control group and PBS control group),6 mice in each group.Mice in each group were immunized with PVAX1-ENO plasmid DNA,PVAX1 empty plasmid and PBS,respectively.Serum was separated and serum ENO protein antibody titers,IgG 1 and IgG 2a antibody levels and IFN-γand IL-4 cytokine levels were determined by ELISA.Two weeks after the third immunization,3 mice from each group were selected to measure the levels of CD4+and CD8+.The results showed that the PVAX1-ENO plasmid DNA was successfully constructed and identified by PCR and restriction endonuclease digestion,and it could be successfully expressed in Vero cells.The level of ENO protein antibody,IgG 1 and IgG 2a antibody,IFN-γand IL-4 cytokines and CD4+and CD8+in PVAX1-ENO plasmid DNA immunized group were significantly higher than those in PVAX1 empty vector control group and PBS control group(P<0.05).The results showed that PVAX1-ENO plasmid DNA could significantly improve the humoral immunity of BALB/c mice.The cellular immune of BALB/c mice was stimulated to some extent.

关 键 词：猪附红细胞体 ENO基因 质粒DNA 细胞免疫 体液免疫 

分 类 号：S858.28[农业科学—临床兽医学]