表达猪附红细胞体ENO基因重组腺病毒的构建及免疫原性分析  

Construction and Immunogenicity Analysis of Recombinant Adenovirus Expressing Mycoplasma suis ENO Gene

作　　者：闫可心 伍生军[1] 赵云 王淼 许应天[1] 薛书江[1] YAN Kexin;WU Shengjun;ZHAO Yun;WANG Miao;XU Yingtian;XUE Shujiang(Agricultural College of Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学农学院

出　　处：《中国畜牧兽医》2019年第7期2088-2095,共8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);吉林省教育厅“十三五”科学技术项目(JJKH20191134KJ)

摘　　要：试验旨在构建表达猪附红细胞体ENO基因的重组腺病毒并分析评价其免疫效果。将重组克隆质粒pMD-19T-ENO与腺病毒穿梭载体AdV4-GFP分别进行双酶切,构建重组腺病毒穿梭质粒AdV4-M/ENO;将经PacⅠ酶线性化后的重组腺病毒穿梭质粒AdV4-M/ENO转染293细胞,获得重组腺病毒Ad4-M/ENO,采用PCR和间接免疫荧光试验(IFTA)鉴定猪附红细胞体ENO基因在293细胞中的表达,再对293细胞进行培养,测定重组腺病毒的滴度;将30只BALB/c小鼠分为3组:重组腺病毒Ad4-M/ENO组、AdV4-GFP空载体对照组和PBS对照组,分别进行免疫接种,采用ELISA方法检测血清中猪附红细胞体IgG、IgG1、IgG2a抗体水平和IFN-γ、IL-4细胞因子水平,在三免2周后检测小鼠脾脏中CD4^+和CD8^+含量。结果显示,构建的重组腺病毒穿梭质粒AdV4-M/ENO目的基因片段大小为1 182 bp;重组腺病毒Ad4-M/ENO包装成功,能在293细胞中表达,滴度为1×109 PFU/mL。经重组腺病毒Ad4-M/ENO免疫后的BALB/c小鼠血清中IgG、IgG1、IgG2a抗体水平,IFN-γ、IL-4细胞因子水平及淋巴细胞亚群CD4^+、CD8^+含量均显著或极显著高于AdV4-GFP空载体对照组和PBS对照组(P<0.05;P<0.01)。结果表明,本试验成功构建了表达猪附红细胞体ENO基因的重组腺病毒,且该重组腺病毒能诱导小鼠产生特异性的体液免疫和细胞免疫应答反应。The aim of the experiment was to construct a recombinant adenovirus expressing Mycoplasma suis ENO gene and analyze its immune effect.Recombinant cloning plasmid pMD-19 T-ENO and adenovirus shuttle vector AdV4-GFP were double digested to construct recombinant adenovirus shuttle plasmid AdV4-M/ENO;The recombinant adenovirus shuttle plasmid AdV4-M/ENO,which was linearized by PacⅠ,was transfected into 293 cells to obtain recombinant adenovirus Ad4-M/ENO.Expression of Mycoplasma suis ENO gene in 293 cells was identified by PCR and indirect immunofluorescence assay(IFTA),293 cells were cultured and the titer of recombinant adenovirus was determined.30 BALB/c mice were divided into three groups:Recombinant adenovirus Ad4-M/ENO group,AdV4-GFP empty vector control group and PBS control group,respectively,vaccinated separately.ELISA was used to detect IgG,IgG1,IgG2 a antibody levels and IFN-γ and IL-4 cytokine levels in serum,and the levels of CD4^+ and CD8^+ in the spleen of mice were detected 2 weeks after the third immunization.The results showed that the recombinant adenoviral shuttle plasmid AdV4-M/ENO gene fragment size was 1 182 bp;The recombinant adenovirus Ad4-M/ENO was successfully packaged and expressed in 293 cells with a titer of 1×109 PFU/mL.The serum levels of IgG,IgG1,IgG2 a,IFN-γ,IL-4 and lymphocyte subsets CD4^+ and CD8^+ in BALB/c mice immunized with recombinant adenovirus Ad4-M/ENO were significantly or extremely significantly higher than AdV4-GFP empty vector control group and PBS control group(P<0.05;P<0.01).The results showed that the recombinant adenovirus expressing Mycoplasma suis ENO gene was successfully constructed in this experiment,and the recombinant adenovirus could induce specific humoral and cellular immune responses in mice.

关 键 词：猪附红细胞体 ENO基因 重组腺病毒 免疫效果 

分 类 号：S858.28[农业科学—临床兽医学]