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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。
Objective To clone and express the gene fragment of the NS3 coding region of Japanese encephalitis virus SA14-14-2 strain, and lay the foundation for the further functional study of recombinant protein. Methods Total RNA was extracted from JEV-SA14-14-2 strain. The NS3-1 and NS3-2 gene fragments were amplified by RT-PCR and cloned into the prokaryotic expression vector pET15bTAT to construct the recombinant prokaryotic expression plasmid. The recombinant plasmid was transformed into E.coli Rosetta2, IPTG induced expression. The expressed product was purified by Ni2 + affinity chromatography and identified by Western blot. Results Recombinant prokaryotic expression plasmids pET15bTAT-NS3-1 and pET15bTAT-NS3-2 were confirmed by enzyme digestion. The recombinant protein expressed relative molecular weight of about 27000 and 20000, mainly in the form of inclusion bodies. Purified recombinant protein purity up to 80%, and can be anti-Japanese encephalitis virus SA14-14-2 serum identification. Conclusion The NS3-1 and NS3-2 genes of Japanese encephalitis virus SA14-14-2 have been successfully cloned and expressed in Escherichia coli Rosetta2.