SARS冠状病毒核衣壳蛋白的重组表达及其单克隆抗体的制备

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 1 269bp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 43 000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 43 000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。

Preparation of specific monoclonal antibody against nucleocapsid protein of SARS coronavirus

AIM: To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein. METHODS: N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. RESULTS: DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the Mr of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as prionary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein. CONCLUSION: The prepared recombinant N protein and mAbs lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.