SARS冠状病毒囊膜E蛋白的重组表达与单克隆抗体制备

Recombination and Expression of SARS Coronavirus Envelop Protein and Generation of Specific Monoclonal Antibody

从基因库中调取SARS冠状病毒囊膜E蛋白基因序列,用连接PCR方法合成完整片段。测序确认后与人免疫球蛋白(IgG)序列Fc段连接并克隆至原核表达载体pPROEXHta中。从SDS-PAGE凝胶中回收表达产物后免疫BALB/c小鼠,经融合、筛选制备特异性单克隆抗体(mAb)。连接PCR扩增出231bp的DNA,测序结果与GenBank公布的序列一致,与人IgG序列Fc段基因连接后克隆至原核表达载体,在大肠杆菌中获得较好表达。表达产物经SDS-PAGE后,在相对分子质量(Mr)约37000处可见1条明显的诱导表达条带。Western印迹的结果表明,该电泳带与SARS患者的恢复期血清呈特异性免疫反应。从SDS-PAGE凝胶中回收表达产物并免疫BALB/c小鼠,制备出2株抗E蛋白的mAb。这些mAb与SDS-PAGE凝胶上Mr约为37000的蛋白带也呈现很强的免疫反应。所获得的重组表达E蛋白及特异性mAb为进一步建立SARS病毒感染早期诊断的方法奠定了基础。

To express small envelop（E） protein of SARS coronavirus and produce monoclonal antibody（mAb） to E protein. E protein gene was amplified bv PCR. After being confirmed by DNA sequencing and linked with human immuno global protein gene Fc segment, the fused gene was sub-cloned into prokaryotic expression vector, pPRO EX Hta. EFc protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. Result of DNA sequencing confirmed that the amplified fragment was E protein gene. SDS-PAGE analysis showed that the expressed protein in E.coli was located at approximately Mr being 37×10^3 on the gel. This protein could be further confinned by Western blot with serum of a SARS patient. The recombinant EFc protein was recovered from SDS-PAGE gel and immunized in BALB/c mice. Western blot analysis proved that two mAbs obtained could reacted specifically to the recombinant EFc protein. The recombinant EFc protein and mAbs lay the foundation for further in development of early diagnosis of SARS coronavirus infection.