口蹄疫病毒受体羊源整联蛋白β6亚基配体结合域的原核表达、纯化及鉴定

摘要

To induce the expression of FMDV receptor integrin β6 subunit ligand-binding domain in prokaryocyte and purify the recombinant protein. The fragment coding ovine integrin β6 ligand-binding domain was amplified by PCR from the recombinant plasmid pGEM-β6. After doubly digested with EcoR I and Sal I ,the β6LBD fragment was subcloned into prokaryotic expression vector pP_(RO)EX~(TM)HTb which was doubly digested with same enzymes.The recombinant expres-sion plasmid pP_(RO)/β6LBD was constructed and transformed into E. coli BL21(DE3) and induced by IPTG.The expres-sion of fusion protein was detected by SDS-PAGE and purified by Ni-NTA His. Bind resins. The expressed product was i-dentified by ELISA and Western blot assay .SDS-PAGE demonstrated that the fusion protein pP_(RO)/β6LBD was expressed efficiently as inclusion body in E. coli with the expected molecular weight of 33 kDa. ELISA and Western blot assays showed that the recombinant protein has the good antigenicity and specificity, which will lay a foundation for further re-search on distribution and the role of the ovine integrin β6 subunit in FMDV infection.%利用原核细胞表达羊口蹄疫病毒(FMDV)受体整联蛋白β6亚基的配体结合域 (LBD)片段,分离纯化目的蛋白.以含有羊整联蛋白β6亚基全长cDNA的质粒为模板,PCR扩增得到β6LBD基因片段,经酶切消化处理后与同样酶切处理的原核表达载体pP_(RO)EX~(TM)HTb相连,构建原核表达质粒pP_(RO)/β6LBD,测序确认读码框正确后,将其转化感受态细胞 BL21 (DE3),IPTG诱导表达重组羊β6LBD融合蛋白.SDS-PAGE鉴定重组蛋白的表达并利用镍离子亲和树脂对其纯化,通过酶联免疫吸附试验(ELISA)和Western blot方法分析鉴定表达产物.成功构建了pPRO/β6LBD原核表达载体,实现了羊FMDV受体整联蛋白亚基β6LBD在大肠杆菌中的高效表达, SDS-PAGE显示其相对分子质量(Mr)约为33 kDa,重组蛋白主要以包涵体形式存在于菌体中.用本试验室保存的抗FMDV受体猪源整联蛋白β6亚基LBD的单克隆抗体进行ELISA 和Western blot检测,显示该单抗可与重组目的蛋白发生特异性反应,证明纯化后的蛋白与特异性抗体具有良好的特异性反应及抗原活性.为深入研究整联蛋白受体β6亚基在羊体内的分布及其在FMDV致病过程中的作用机制奠定了基础.