乙型脑炎病毒NS5蛋白的表达、纯化及多克隆抗体的制备

目的表达纯化乙型脑炎病毒非结构蛋白NS5,并制备其多克隆抗体。方法通过PCR法扩增编码NS5蛋白的全长基因,将其克隆到原核表达载体pET28a中,转化大肠杆菌BL21（DE3）进行诱导表达,并通过Ni柱亲和层析纯化重组蛋白。用纯化后的重组蛋白免疫BALB/c鼠制备多克隆抗体。采用间接免疫荧光法检测抗体效价,Western印迹鉴定抗体的特异性。结果与结论成功获得了可溶性表达的NS5蛋白,制备了多克隆抗体,效价为1∶1000,能够特异性识别乙型脑炎病毒感染细胞中表达的NS5蛋白,为进一步研究NS5蛋白的生物学功能奠定了基础。

Expression and purification of nonstructural protein 5 of Japanese encephalitis virus and preparation of its polyclonal antibody

Objective To express and purify the nonstructural protein NS5 of Japanese encephalitis virus（JEV）and prepare the corresponding polyclonal antibodies.Methods The full length sequence encoding the NS5 protein was amplified by PCR and subsequently cloned into the prokaryotic expression vector of pET28a.The recombinant His-NS5 fusion protein was expressed in E.coli BL21（DE3）and purified by the Ni-agarose His tag affinity chromatography.Then the anti-NS5 antiserum was prepared in mice.The antibody titer was determined by immunofluorescence assay,and the specificity was identified by Western blot assay.Results and Conclusion The NS5 protein of JEV was successfully expressed in a soluble form and the polyclonal antibodies against it were prepared accordingly.The antibody titer was about 1∶1000,and NS5 expressed in the cells infected by JEV could be recognized by this antiserum.