【摘要】 目的克隆并表达乙型脑炎病毒SA14-14-2株NS3编码区的基因片段,为重组蛋白进一步的功能研究奠定基础。方法提取乙脑病毒SA14-14-2株总RNA,用RT-PCR法扩增NS3-1、NS3-2基因片段,克隆入原核表达载体pET15bTAT中,构建重组原核表达质粒,转化大肠杆菌Rosetta2,IPTG诱导表达。表达产物经Ni2+亲和层析柱纯化后,进行Western blot鉴定。结果重组原核表达质粒pET15bTAT-NS3-1和pET15bTAT-NS3-2经酶切证明构建正确。表达的重组蛋白相对分子质量约为27000和20000,主要以包涵体的形式表达。重组蛋白纯化后纯度可达80%以上,并可被小鼠抗乙型脑炎病毒SA14-14-2株血清识别。结论已成功克隆了乙型脑炎病毒SA14-14-2株NS3-1和NS3-2基因,并在大肠杆菌Rosetta2中获得表达。更多还原

【Abstract】 Objective To clone and express the gene fragment encoding NS3 of Japanese encephalitis(JE)virus SA14-14-2 strain and lay a foundation of further study on the function of recombinant NS3 protein.Methods The total RNA of JE virus SA14-14-2 strain was extracted for amplification of NS3-1 and NS3-2 gene fragments by RT-PCR.The amplified gene fragments were cloned into prokaryotic expression vector pET15bTAT respectively,and the constructed recombinant plasmids were transformed to E.coli Rosetta2 for expression under induction of IPTG.The expressed products were purified by nickel ion affinity chromatography and identified by Western blot.Results Restriction analysis proved that recombinant plasmids pET15bTAT-NS3-1 and pET15bTAT-NS3-2 were constructed correctly.The expressed recombinant proteins,with relative molecular masses of 27 000 and 20 000 respectively,mainly existed in forms of inclusion bodies,reached purities of more than 80%and were recognized by the murine antisera against JE virus SA14-14-2 strain.Conclusion The NS3-1 and NS3-2 genes of JE virus SA14-14-2 strain were successfully cloned and expressed in E.coli Rosetta2.更多还原