【摘要】 【目的】利用原核表达系统表达鸭坦布苏病毒(Duck Tembusu virus,DTMUV)的NS3蛋白,并以此制备鼠抗NS3蛋白的多克隆抗血清。【方法】利用PCR扩增鸭坦布苏病毒CQW1株NS3基因片段,然后将其克隆到pET-28a(+)载体,构建重组质粒pET-28a(+)-NS3。将重组表达质粒转化到大肠杆菌BL21感受态细胞中,筛选获得阳性重组菌,经IPTG诱导表达,通过SDS-PAGE和Western blot试验表明重组NS3蛋白在BL21细胞中成功表达,将重组NS3蛋白切胶纯化后免疫小鼠后收获抗血清。【结果】经间接免疫荧光试验和Western blot试验验证,多克隆抗血清可识别病毒感染细胞过程产生的NS3蛋白和真核表达的NS3蛋白,具有良好的反应性和特异性。【结论】成功制备的DTMUV NS3蛋白的鼠源多克隆血清,将为鸭坦布苏病毒的临床检测和NS3蛋白功能性研究奠定基础。更多还原

【Abstract】 【Objective】In this study,NS3 protein of Duck Tembusu virus(DTMUV) was expressed usingprokaryotic expression system,and subsequently used to prepare polyclonal anti-serum of mouse antiNS3 protein.【Method】NS3 gene fragment of CQW1 strain of DTMUV was amplified by PCR,and then cloned into p ET-28 a(+) vector to construct recombinant plasmid pET-28 a(+)-NS3. The recombinant expression plasmid was transformed into Escherichia coli BL21 competent cells,and the positive recombinant bacteria were screened and expressed by IPTG induction. The results based on SDS-PAGE and Western blot showed that the recombinant NS3 protein was successfully expressed in BL21 cells. The recombinant NS3 protein was purified and used to immunize mice,the antiserum was harvested.【Result】The indirect immunofluorescence assay and Western blot assay confirmed that the polyclonal antiserum can recognize the NS3 protein produced by the DTMUV-infected cells and the NS3 protein expressed by eukaryotic cells,which has good reactivity and specificity.【Conclusion】The mouse polyclonal sera of DTMUV NS3 protein successfully prepared in this study,and it will lay the foundation for the clinical detection of DTMUV and the functional study of NS3 protein.更多还原