牛源犬新孢子虫NcGRA9基因原核表达及免疫原性分析  

Prokaryotic expression and immunogenicity analysis of recombinant NcGRA9 protein of bovine Neospora caninum

作　　者：金卫东 唐泽宇 李作臣 王海军 陈健 贾立军[1] JIN Weidong;TANG Zeyu;LI Zuochen;WANG Haijun;CHEN Jian;JIA Lijun(Engineering Research Center of North-East Cold Region Beef Cattle Science&Technology Innovation of Ministry of Education,Yanbian University,Yanji,Jilin 133002,China;Characteristic Industry Development Center of Yanbian,Yanji,Jilin 133002,China;Animal Husbandry Station of Yanbian Autonomous Prefecture,Yanji,Jilin 133002,China;Animal Disease Control Center of Yanbian Autonomous Prefecture,Yanji,Jilin 133002,China)

机构地区：[1]延边大学东北寒区肉牛科技创新教育部工程研究中心,吉林延吉133002 [2]延边特色产业发展中心,吉林延吉133002 [3]延边州畜牧总站,吉林延吉133002 [4]延边州动物疫病预防控制中心,吉林延吉133002

出　　处：《中国兽医学报》2023年第2期303-306,347,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32160839);吉林省中青年科技创新领军人才及团队资助项目(20200301034RQ);高等学校学科创新引智“111”计划资助项目(D20034);延边大学科研创新团队资助项目(延大校发[2018]203号)。

摘　　要：为了确定牛源犬新孢子虫NcGRA9基因的功能及表达蛋白的免疫原性,本试验PCR扩增牛源犬新孢子虫NcGRA9基因,构建克隆质粒pMD18-T-NcGRA9和原核表达质粒pGEX-4T-NcGRA9,转化大肠杆菌BL21感受态细胞中IPTG诱导表达,SDS-PAGE电泳和Western blot分析表达重组蛋白的反应原性,应用重组蛋白与弗氏佐剂混合接种BALB/c小鼠,ELISA检测小鼠血清中IgG、IgG1、IgG2a抗体水平以及IFN-γ、IL-4细胞因子水平。结果显示,经PCR扩增获得522 bp的NcGRA9片段,表达蛋白纯化后相对分子质量约为45 kDa,具有良好的反应原性;重组蛋白接种BALB/c小鼠后,免疫组小鼠IgG、IgG1、IgG2a抗体水平以及IFN-γ、IL-4细胞因子水平均极显著高于PBS对照组(P<0.01),说明表达的重组蛋白具有良好的免疫原性。本试验表达的NcGRA9重组蛋白具有良好的抗原性,为新孢子虫病的防控奠定了基础。To determine the function and immunogenicity of GRA9 gene of Neospora caninum,NcGRA9 gene was amplified by PCR,and then the plasmid pMD18-T-NcGRA9 and prokaryotic expression plasmid pGEX-4T-NcGRA9 were constructed.The expression of recombinant NcGRA9(rNcGRA9)was induced by IPTG after pGEX-4T-NcGRA9 plasmid was transferred into E.coli BL21 competent cells.rNcGRA9 was confirmed by SDS-PAGE and Western blot.Purified rNcGRA9 was used to inoculate BALB/c mice with Freund’s adjuvant.The levels of IgG,IgG1,IgG2a,IFN-γ and IL-4 in mouse serum were detected by ELISA.The results showed that the NcGRA9 fragment was 522 bp.The molecular weight of the rNcGRA9 was about 45 kDa,and it had good reactivity.After BALB/c mice were immunized with recombinant protein,the antibody levels of IgG,IgG1,IgG2a and IFN-γ were increased,and the level of IL-4 cytokine was significantly higher than those in the control group(P<0.01),indicating that the rNcGRA9 had good immunogenicity.In summary,the rNcGRA9 subunit vaccine with clear immunogenicity was prepared successfully,which laid a foundation for the prevention and control of neosporosis.

关 键 词：犬新孢子虫 NcGRA9 原核表达 重组蛋白 免疫原性 

分 类 号：S852.62[农业科学—基础兽医学]