摘要
研究旨在利用哺乳动物细胞悬浮培养系统表达非洲猪瘟病毒(ASFV)p17蛋白,纯化并免疫小鼠,制备针对ASFV p17蛋白的特异性多克隆抗体。根据GenBank中公布的ASFV SY18毒株p17蛋白编码基因序列,设计特异性引物扩增p17基因片段,构建重组真核表达质粒pCDNA3.1-p17-strep。将其瞬时转染293i细胞,并利用下游strep标签进行蛋白纯化。纯化后的重组p17蛋白配合MnJ(β)胶体锰佐剂免疫BALB/c小鼠制备多克隆抗体血清。利用Western blot、间接免疫荧光试验鉴定该多克隆抗体的反应原性和特异性。结果显示:本试验成功构建pCDNA3.1-p17-strep真核表达质粒,转染293i细胞后纯化获得重组p17蛋白。免疫小鼠后制备的多克隆抗体与真核表达的p17蛋白及表达ASFV p17蛋白的猪繁殖与呼吸综合征病毒均有良好的特异性免疫反应。本试验为深入探讨ASFV p17蛋白的生物学功能奠定了基础。
The aim of this study was to express p17 protein of African swine fever virus(ASFV) in mammalian cells using the suspension culture system, and then the recombinant protein was purified and administered into mice for production of polyclonal antibodies. The recombinant eukaryotic expression plasmid pCDNA3.1-p17-strep was constructed by amplifying the p17 gene fragment based on the ASFV SY18 strain. The recombinant protein was used to immunize Balb/c mice with colloidal manganese adjuvant to prepare polyclonal antibody. The reactogenicity and specificity of the antibody were analyzed by Western blot and indirect immunofluorescence. The results showed that the pCDNA3.1-p17-strep eukaryotic expression plasmid was successfully constructed and transfected into 293i cells. The prepared polyclonal antibody specifically reacted with the p17 protein expressed in the cells and with the recombinant porcine reproductive and respiratory syndrome virus expressing p17 gene. This study laid a basis for the in-depth exploration of the biological function of ASFV p17 protein.
作者
乔思娜
赵款
刘佳晨
李国新
童武
周艳君
赵冉
童光志
高飞
李丽薇
QIAO Sina;ZHAO Kuan;LIU Jiachen;LI Guoxin;TONG Wu;ZHOU Yanjun;ZHAO Ran;TONG Guangzhi;GAO Fei;LI Liwei(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China;Xiamen Center for Animal Disease Control and Prevention,Xiamen 361009,China)
出处
《畜牧与兽医》
CAS
北大核心
2023年第3期68-73,共6页
Animal Husbandry & Veterinary Medicine
基金
上海市科技兴农项目(2022-02-08-00-12-F01162)
广东省重点领域研发计划(2019B020211003)
国家自然科学基金ASFV专项(31941017)
中国农科院统筹ASFV应急专项(Y2020YJ15)
河北省省属高等学校基本科研业务费(KY202017)。