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42000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∶12800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。
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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第10期975-978,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展计划(973)资助项目(2005CB523201)
国家支撑计划资助项目(2006BAD06A14)
