口蹄疫病毒受体猪源整联蛋白β6亚基配体结合域多克隆抗体制备.docVIP

口蹄疫病毒受体猪源整联蛋白β6亚基配体结合域多克隆抗体制备 　 作者：高闪电, 常惠芸, 独军政, 王景锋, 周建华, 赵建勇, 谢庆阁 【摘要】 目的: 表达口蹄疫病毒受体猪源整联蛋白β6亚基配体结合域β6LBD, 并制备兔抗β6LBD多克隆抗体。方法: 利用PCR方法扩增整联蛋白β6LBD基因片段, 经BamHⅠ和XhoⅠ进行双酶切后连入pGEX4T1原核表达载体, 构建的 pGEX4T1β6LBD重组表达质粒, 转化大肠杆菌BL21(DE3)菌株, 经IPTG诱导表达, SDSPAGE鉴定融合蛋白的表达并提取包涵体, 纯化目的蛋白。然后用纯化的蛋白免疫新西兰大耳白兔制备其多克隆抗体, 以ELISA方法测定抗体效价, 并以Western blot鉴定其特异性。结果: SDSPAGE电泳分析显示pGEX4T1β6LBD诱导后表达一Mr约为42 000的GSTβ6LBD融合蛋白, 与预期结果相符。目的蛋白纯化后, 在电泳图片上显示较为清晰的单一条带, 用纯化GSTβ6LBD免疫新西兰大耳白兔制备的多克隆抗体效价达1∶12 800以上, 具有较高的特异性。结论: 制备了具有高亲和性和特异性的抗猪源整联蛋白β6LBD多克隆抗体, 为深入研究整联蛋白β6在口蹄疫病毒感染过程中的作用了奠定基础。 【关键词】 口蹄疫病毒; 受体; 整联蛋白β6亚基; 配体结合域; 多克隆抗体 ［Abstract］ AIM: To induce the expression of FMDV receptor integrin β6 subunit ligandbinding domain in E.coli and prepare the rabbit polyclonal antibody against it. METHODS: The fragment coding β6 ligandbinding domain was amplified by PCR and doubly digested with BamH Ⅰand Xho Ⅰ. Then it was cloned into expression vector pGEX4T1 to obtain recombinant plasmid pGEX4T1β6LBD. After pGEX4T1β6LBD was transformed into E.coli BL21(DE3) and induced by IPTG, the expression of fusion proteins was identified by SDSPAGE, with inclusion body prepared and fusion protein purified. Then new Zealand rabbits were immunized to prepare polyclonal antibody against β6LBD. GSTβ6LBD antiserum was obtained and the specificity of polyclonal antibody was detected by Western blot. RESULTS: SDSPAGE demonstrated that the fusion protein GSTβ6LBD was expressed with the expected molecular weight at 42 000. A single clear band of GSTβ6LBD fusion protein appeared in SDSPAGE gel after purification. The titer of the polyclonal antibody was above 1∶12 800 and it is of high specificity. CONCLUSION: The successful preparation of rabbit anti pig β6LBD polyclonal antibody with high affinity and specificity will lay a foundation for further research into the function of integrin β6 in FMDV infection. ［Keywords］FMDV; receptor; integrin β6 subunit; ligandbinding domain; polyclonal antibo