鸭疫里氏杆菌OmpA基因真核表达载体构建及免疫效果初步评价  

Construction of an eukaryotic expression vector of ompA gene of Riemerella anatipestifer and preliminary evaluation of its immune effect

作　　者：余波 李婷 徐景峨 张亚楠 姜玲玲 周思旋 卜仕金[1] YU Bo;LI Ting;XU Jing’e;ZHANG Yanan;JIANG Lingling;ZHOU Sixuan;BU Shijin(College of Veterinary Medicine,Yangzhou University/Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infections Diseases and Zoonoses,Yangzhou 225009,China;Institute of Animal Husbandry and Veterinary Medicine of GAAS,Guiyang 550005,China)

机构地区：[1]扬州大学兽医学院/江苏省动物重要疫病与人畜共患病防控协同创新中心,江苏扬州225009 [2]贵州省农科院畜牧兽医研究所,贵州贵阳550005

出　　处：《畜牧与兽医》2021年第3期84-88,共5页Animal Husbandry & Veterinary Medicine

基　　金：江苏高校优势学科建设工程资助项目,贵州省农科院创新专项(黔农科院科技创新[2017]03号);贵州省科技计划项目(黔科合支撑[2019]2287号);贵州省科技计划项目(黔科合平台人才[2019]5203号);国家水禽产业技术体系贵阳综合试验站(CARS-42-53)。

摘　　要：为构建鸭疫里氏杆菌外膜蛋白A(OmpA)基因真核表达重组质粒,同时验证其在DF-1细胞中的表达和对雏鸭的免疫保护效果,以pVAX1为真核表达载体,构建真核表达质粒pVAX1-OmpA,通过质粒PCR、双酶切和测序进行鉴定,将鉴定后的阳性质粒pVAX1-OmpA转染至DF-1细胞,采用间接免疫荧光试验(IFA)和Western blot方法检测OmpA基因在DF-1细胞中的表达;将pVAX1-OmpA DNA免疫雏鸭,采集血清检测免疫抗体,并进行攻毒保护试验。结果,成功构建真核表达质粒pVAX1-OmpA,两种免疫学方法均检测到ompA蛋白的特异性表达,免疫雏鸭后14、28、35、49、63 d抗体水平与灭活疫苗组差异不显著,对雏鸭的免疫保护率达到100%。结果表明,pVAX1-OmpA重组质粒免疫雏鸭后能够诱导产生特异性的免疫抗体,能够产生较强的免疫保护效果,可作为新型DNA疫苗的候选。In order to construct an eukaryotic expression recombinant plasmid of the outer membrane protein gene of Riemerella anatipestifer,and to verify its expression in DF-1 cells and its immunoprotective effect on ducklings,pVAX1 was used as an eukaryotic expression vector to construct an eukaryotic expression plasmid pVAX1 OmpA which was identified by plasmid PCR,double enzyme digestion and sequencing.The identified positive eukaryotic expression plasmid pVAX1-OmpA was then transfected into DF-1 cells.IFA and Western blot were used to detect the transcription of the ompA protein;pVAX1-OmpA DNA was used to immunize ducklings from whom serum samples were collected for detection of immune antibody,and finally a protection test was carried out.The results showed that the eukaryotic expression plasmid pVAX1 OmpA was successfully constructed,the expression of the OmpA protein was detected by IFA and Western blot.There was no significant difference in the antibody levels between the recombinant plasmid pVAX1-OmpA group and the inactivated vaccine group on 14,28,35,49 and 63 days after immunization,and the immune protection rate of the ducklings reached 100%.The above results indicated that the recombinant plasmid pVAX1-OmpA induced specific immune antibodies in ducklings,and produced a strong immune protection effect.The recombinant plasmid might be used as a candidate strain of a new DNA vaccine.

关 键 词：鸭疫里氏杆菌 OmpA基因 真核表达 免疫保护效果 DNA疫苗 

分 类 号：S852.61[农业科学—基础兽医学]