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。
Objective To verify the immune responses induced by the recombinant membrane glycoprotein （M protein） of SARS eoronavirus in mice. Methods The gene fragment of the M protein was amplified by PCR and was subcloned into prokaryotlc expression vector pET-23a. The recombinant plasmld pETM 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 SDSPAGE 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 ： 104, 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-y 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.
出处
《中国病原生物学杂志》
CSCD
2006年第5期331-334,共4页
Journal of Pathogen Biology
基金
深圳市科技计划项目(No.2003-k-07)
关键词
SARS冠状病毒
M蛋白
免疫效应
SARS eoronavirus
membrane glycoprotein
immune response
