摘要
首先,通过PCR扩增和同源重组技术构建了pET-28a-H124R重组原核表达质粒。经IPTG诱导,pH124R蛋白在大肠杆菌中高效表达,并通过镍柱亲和层析法纯化获得重组蛋白。随后,用pH124R重组蛋白免疫豚鼠,制备了特异性多克隆抗体。通过SDS-PAGE、Western-blot和间接免疫荧光试验(IFA)对其进行检测,结果表明该抗体能够特异性识别ASFV感染细胞中表达的内源pH124R蛋白,且该蛋白主要定位于细胞质病毒工厂区域。本研究成功实现了ASFV pH124R蛋白的原核表达,并制备了可用于Western-blot和IFA试验的特异性多克隆抗体,为深入研究pH124R蛋白的生物学功能提供了重要生物学材料。
First,the pET-28a-H124R recombinant prokaryotic expression plasmid was constructed using PCR amplification and homologous recombination techniques.Following IPTG induction,the pH124R protein was efficiently expressed in E.coli and purified by Ni-NAT affinity chromatography to obtain the recombinant protein.Subsequently,guinea pigs were immunized with the purified pH124R recombinant protein to prepare specific polyclonal antibodies.Detection by SDS-PAGE,Western-blot,and indirect immunofluorescence assay(IFA)showed that the antibody specifically recognizes the endogenous pH124R protein expressed in ASFV-infected cells,with the protein primarily localized in the cytoplasmic viral factory regions.This study successfully achieved the prokaryotic expression of the ASFV pH124R protein and prepared specific polyclonal antibodies suitable for Western-blot and IFA experiments.These antibodies provide important biological materials for in-depth studies on the biological functions of the pH124Rprotein forAfrican swine fever.
作者
戴观丽
周艳龙
武正前
宋大铭
蔡祎平
周玉龙
李丹
郑海学
DAI Guanli;ZHOU Yanlong;WU Zhengqian;SONG Daming;CAI Yiping;ZHOU Yulong;LI Dan;ZHENG Haixue(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gulang County Agricultural Industrialization Service Center,Weiwu 733000,China)
出处
《中国兽医科学》
北大核心
2025年第7期925-931,共7页
Chinese Veterinary Science
基金
甘肃省重大人才项目(212068657016)
甘肃省陇原青年英才(20230102)
甘肃省科技重大专项(21ZD3NA001-5,22ZD6NA001)
国家“十四五”重点研发计划项目(2021YFD1800100)
黑龙江省“双一流”学科协同创新成果项目(LJGXCG2024-F07)
甘肃省青年科技基金项目(25JRRA445)。
关键词
非洲猪瘟病毒
pH124R蛋白
原核表达
多克隆抗体
African swine fever virus
pH124R protein
prokaryotic expression
polyclonal antibodies
作者简介
戴观丽(1999-),女,广西梧州人,硕士生,专业为预防兽医学,E-mail:gl_dai1990@163.com;通讯作者:周玉龙(1980-),男,教授,博士生导师,主要从事动物分子病毒学研究,E-mail:zhouyulong@163.com;通讯作者:李丹(1982-),男,研究员,博士生导师,主要从事新发病的致病、感染和免疫机制的研究,E-mail:lidan@caas.cn;通讯作者:郑海学(1979-),男,研究员,博士生导师,主要从事动物传染病学与流行病学研究,E-mail:zhenghaixue@caas.cn。
