口服免疫Ag85A脂质体DNA诱导T细胞亚群应答效应研究

Research on the induction of T cell subset response via oral vaccination with liposome-mediated recombinant pCDNA3.1^+/Ag85A plasmid

目的:拟采用构建的可在真核细胞内表达Ag85A基因的质粒,以阳离子脂质体为运载体,制成DNA疫苗,经口途径投予小鼠,以观察Ag85A脂质体DNA疫苗诱导免疫应答效应,为口服DNA疫苗的临床应用提供理论和实验依据。方法:ELISA方法检测Ag85A特异性抗体产生水平及血清Th1型细胞因子IFN-γ及Th2型细胞因子IL-4的分泌水平,ELISPOT技术检测口服DNA疫苗后小鼠可分泌IFN-γ和IL-4脾淋巴细胞数量。流式细胞术观察口服DNA疫苗后小鼠脾淋巴细胞CD4+ T细胞及CD8+ T细胞亚群的变化,从而判断口服DNA疫苗的免疫效果及脂质体是否有免疫增强作用。结果:口服自制Ag85A DNA疫苗可见血清中抗Ag85A特异性抗体的产生;下调了脾CD4+ T细胞和CD8+T细胞亚群的数量;分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降,而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。口服DNA疫苗组诱导Ag85A特异性抗体产生,脂质体具有免疫佐剂作用。结论:应用阳离子脂质体为运载体,重组Ag85A DNA疫苗口服免疫C57BL/6小鼠,诱导了CD4+及CD8+ T细胞亚群的下调及IFN-γ的表达减少,而IL-4的分泌呈增高趋势;疫苗可诱导Ag85A特异性抗体的分泌,产生了明显的体液应答。

Objective：To prepare recombinant pCDNA3.1 ＋/Ag85A DNA vaccine encoding tuberculosis Ag85A gene mediated with LipofectamineTM 2000 and to study the effect on T cell subpopulation via oral vaccination. Methods： Recombinant plasmid containing Ag85A using liposome as a vector was constructed and administered to C57BL/6 mice via oral route. Determination of the contents of IFN-γ, IL-4 in serum of C57BL/6 mice was performed by double antibody sandwich ELISA. Furthermore, the ability of splenocytes to secret IFN-γ and IL-4 was tested by ELISPOT method. When the mice were vaccinated with recombinant eukaryotic expressing vector 5 weeks later, titers of serum antibody against Ag85A were detected by ELISA. The percentage of the CD4^＋ and CD8 ^＋ T cell subsets in the splenocytes was determined by flow cytometry. Results： Lymphocytes obtained from the spleen of liposomal pcDNA3, 1^＋/Ag85A vaccine-immunized mice exhibited lower IFN-γ/production and higher IL-4 production than those of pcDNA3.1^＋ vector immunized mice. The number of spleen MNC secreting IFN-γ stimulated by Ag85A protein in vitro was significantly lower than that of plasmid vector group. Liposomal pcDNA3.1＋/AgSJA vaccine immunized mice elicited higher Ag85A-specific antibodcy titres. The percentage of the CD4 ＋ and CD8＋ T cell subsets in the splenocytes was decreased. The subset both were shown the profile of Th2 responses. Conclusion：The oral recombinant plasmid pCDNA3.1＋/Ag85A mediated with LipofectamineTM 2000 It shows down-regulation effect on the subsets of CD4＋ T cells and CD8＋T cells. The regimen has good immunogenecity and could induce Th2 type humoral response in C57BL/6 mice. The immunization suppresses secretion of IFN-γ. It can greatly enhance the fitres of Ag85A-specific antibodies. LipofectamineTM 2000 can act as an adjuvant through comparation with negative control group.