【摘要】 目的检测SARS冠状病毒重组M蛋白疫苗诱导小鼠的免疫应答能力。方法PCR扩增M蛋白基因,构建至原核表达质粒pET-23a,pET-23a-M重组质粒转化BL21(DE3)宿主菌,诱导表达蛋白经SDS-PAGE、免疫印迹分析和纯化后免疫BALB/c小鼠,3次免疫后测定免疫功能,进行免疫效果评价。结果成功构建pET-23a-M重组质粒,转化宿主菌后诱导表达27 ku M蛋白。检测M蛋白免疫鼠体内有特异性抗体产生,抗体效价达1∶104;脾脏CD8+比例升高,CD4+/CD8+比值下降;免疫鼠血清IFN-γ和IL-4水平无显著变化。结论SARS病毒重组M蛋白疫苗能诱导BALB/c小鼠产生特异性体液免疫和细胞免疫应答。更多还原

【Abstract】 Objective To verify the immune responses induced by the recombinant membrane glycoprotein(M protein) of SARS coronavirus in mice.Methods The gene fragment of the M protein was amplified by PCR and was subcloned into prokaryotic expression vector pET-23a.The recombinant plasmid pET-M was then transformed into E.coli BL21(DE3),and the positive clone was induced with IPTG to express the target protein M.After being characterized by SDS-PAGE and Western blot,the recombinant protein was purified from E.coli cell lysate by metal-chelating chromatography.The immunological activities of this protein were evaluated by detection of immune responses in mice inoculated with the recombinant M protein for three times.Results It was demonstrated that the pET-M was constructed through subcloning the right insert of M protein into pET-23a,and the 27 ku recombinant protein was expressed in clone containing pET-M when induced with IPTG.The purified M protein could induce specific IgG antibody in immunized mice,which the titer reached 1∶10~4.The percentage of CD8~+ T lymphocyte of the spleen increased dramatically while the CD4~+/CD8~+ ratio decreased significantly when compared with mice control. No significant difference of IFN-γ and IL-4 concentration in sera were watched between the immunize and control mice.Conclusion It is concluded that the recombinant M protein of SARS coronavirus could induce both specific humoral and cellular immune response in BALB/c mice.更多还原