博尔纳病毒核蛋白的原核表达、纯化及鉴定

Expression,purification and assessment of Borna disease virus nucleoproteinin Escherichia coli

目的构建、表达、并纯化重组博尔纳病毒核蛋白(Borna disease virus,BDV),并鉴定该蛋白。方法通过PCR反应从博尔纳病毒cDNA中扩增博尔纳病毒核蛋白P40基因,构建博尔纳病毒核蛋白P40基因重组质粒pET-14b-BD-VP40,转化大肠杆菌,IPTG诱导融合蛋白的表达,His-tag亲和层析纯化该蛋白,SDS-PAGE分析其表达量、表达形式和纯度;Western-blot法鉴定该蛋白。结果成功构建重组质粒pET-14b-BDVP40,酶切鉴定、核酸序列分析正确,亲和层析纯化后纯度可达90%,Western-blot表明重组蛋白能与博尔纳病毒核蛋白单克隆抗体特异性结合。结论在大肠杆菌中成功表达了可溶性的pET-14b-BDVP40融合蛋白,为进一步研究博尔纳病毒核蛋白作用机制及开发相应血清学检测试剂盒提供基础。

Objective To express and purify recombinant Borna disease virus nucleoprotein in E.coli,analyzing it by specific monoclonal antibodies.Methods Construction expression vector pET-14b-BDVP40 containing the recombinant Borna disease virus nucleoprotein gene.The constructed vector which had been identified by enzyme digestion and nucleotide sequence analysis was transformed into E.coli BL21(DE3).After induced with IPTG,the recombinant protein was purified with His affinity chromatography and evaluated its purit...