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  

Prokaryotic Expression,Purification and Identification of Ligand Binding Domain of Ovine Integrin β6 as FMDV Receptor

作　　者：薛霜[1,2] 独军政[1] 高闪电[1] 常惠芸[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,国家口蹄疫参考实验室,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,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《华北农学报》2009年第5期55-58,共4页Acta Agriculturae Boreali-Sinica

基　　金：国家“863”项目(2006AA10A204);国家支撑计划资助项目(2006BAD06A14)

摘　　要：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pPROEXTMHTb相连,构建原核表达质粒pPRO/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。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 pPROEXTMHTb which was doubly digested with same enzymes.The recombinant expression plasmid pPRO/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 PPRo/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 research on distribution and the role of the ovine integrin 136 subunit in FMDV infection.

关 键 词：口蹄疫病毒 受体 整联蛋白β6亚基 配体结合域 原核表达 

分 类 号：Q785[生物学—分子生物学]