猪口蹄疫病毒受体整合素β8亚基配体结合域的原核表达和多克隆抗体制备  

Prokaryotic expression of ligand binding domain of porcine integrin β8 as FMDV receptor and preparation of its polyclonal antibody

作　　者：独军政[1] 高闪电[1] 常惠芸[1] 赵建勇[1] 丛国正[1] 邵军军[1] 林彤[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2008年第3期8-12,共5页Journal of Gansu Agricultural University

基　　金：国家支撑计划项目(2006BAD06A03);国家高技术研究发展计划(863)项目(2006AA10A204)

摘　　要：病毒受体是决定病毒宿主特异性和组织嗜性的重要因素.利用原核细胞表达了猪口蹄疫病毒受体整合素β8亚基的配体结合域(LBD)片段,分离纯化目的蛋白后免疫兔,制备兔多克隆抗体.以含有猪β8亚基全长cD-NA的质粒为模板,经PCR扩增得到位于β8成熟肽氨基端的LBD基因片段,与带有GST标签的原核表达载体pGEX 4T-1相连,构建原核表达质粒pGEX/β8LBD,测序确认读码框正确后,将其转化感受态细胞BL21(DE3),以IPTG诱导表达重组β8LBD融合蛋白.通过SDS-PAGE和Western blot鉴定后提取包涵体,以电泳分离纯化的重组融合蛋白免疫新西兰白兔制备多克隆抗体,鉴定血清效价和特异性.结果显示,实现了重组猪β8LBD融合蛋白在大肠杆菌中的高效表达,SDS-PAGE显示分子量约为46 000,主要以包涵体形式存在于菌体中.制备的兔多克隆抗体效价达1∶12 800以上,该血清可与目的蛋白发生特异性反应.这将为进一步研究猪整合素β8亚基在宿主体内的分布及其在口蹄疫病毒致病过程中的作用奠定基础.Receptors play a crucial role in determining the pathogenesis and tissue tropism of virus.In this study,we expressed the ligand binding domain（LBD） of porcine integrin β8 as foot-and-mouth disease virus（FMDV）receptor in E.coli and prepared its polyclonal antibody.The LBD fragment of porcine integrinβ8 gene was obtained by PCR from the recombinant plasmid pGEM-β8.The LBD fragment was subcloned into prokaryotic expression vector pGEX 4T-1.The recombinant expression plasmid pGEX/β8LBD was constructed and transformed into competent E.coli BL21（DE3）.The recombinant porcine integrinβ8LBD protein was expressed after IPTG induction and purified from total protein of BL21（DE3） transformed by the plasmid pGEX/β8LBD.The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antibody.The antibody was identified by Western blot and ELISA.The results showed that the recombinant porcine integrinβ8LBD protein was expressed efficiently as inclusion body in E.coli with a molecular weight of approximate 46 000.The titer of the polyclonal antibody against the integrinβ8LBD protein was about 1∶12 800 by ELISA.The expression and purification of porcine integrinβ8LBD protein and the preparation of its polyclonal antibody lay a foundation for understanding the interaction of FMDV with porcine integrinβ8 subunit.

关 键 词：猪 口蹄疫病毒受体 β8亚基配体 结合域 原核表达 多克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学] Q78[农业科学—兽医学]