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目的:表达SARS冠状病毒核衣壳蛋白(N蛋白),并以表达产物为免疫原制备特异性单克隆抗体(mAb).方法:采用RT PCR方法,从灭活的病毒抗原标本中扩增编码N蛋白的基因.测序确认后,再亚克隆至原核表达载体中.从SDS PAGE凝胶中 回收原核表达产物后免疫BALB/c小鼠,经融合、筛选制备特异性mAb.结果:从标本中扩增出1269bp的DNA,测序结果证实 为N蛋白基因.将该基因克隆至原核表达载体中后,在大肠杆菌中获得了较好的表达.表达产物经SDS PAGE后,在Mr约为 43000处可见1条明显的诱导表达带.Westernblot的结果表明,该电泳带与SARS患者的恢复期血清呈特异的免疫反应.从 SDS PAGE凝胶中回收表达产物并免疫BALB/c小鼠,制备出3株抗N蛋白的mAb.这些mAb与SDS PAGE凝胶上Mr约为 43000的蛋白带也呈现很强的免疫反应.结论:所获的重组N蛋白及特异性mAb,将为进一步建立SARS病毒感染早期诊断的 方法奠定了基础.
Objective: To express the SARS coronavirus nucleocapsid protein (N protein) and to use the expressed product as immunogen to prepare specific monoclonal antibody (mAb) .Methods: RT-PCR was used to amplify the coding sequence of inactivated virus antigen N protein.The sequencing was confirmed and then subcloned into the prokaryotic expression vector.After the prokaryotic expression product was recovered from the SDS PAGE gel, the BALB / c mice were immunized and fused and screened to prepare the specific mAb.Results: 1269bp DNA was amplified and sequenced as N protein.The gene was cloned into prokaryotic expression vector and expressed in E.coli.After SDS PAGE, A clear induction of the expression of the band can be seen.Westernblot results showed that the electrophoresis zone and SARS patients convalescent sera specific immune response was recovered from SDS PAGE gel and immunized BALB / c mice prepared 3 These mAbs showed a strong immune response with the protein band of Mr about 43000 on SDS PAGE gel.Conclusion: The obtained recombinant N protein and specific mAb will provide a new way to further establish SARS virus infection The method of early diagnosis has been laid Foundation.