新型抗结核蛋白亚单位疫苗Ag85A-γ干扰素的免疫效果评价

Evaluation of the immunological effects of Ag85A-interferon-γ fusion protein on C57BL/6 mouse

本研究旨在评价新型抗结核融合蛋白亚单位疫苗Ag85A-γ干扰素(Ag85A-IFN-γ)的免疫效果。在成功表达并纯化了去除Ag85A信号肽的融合蛋白Ag85A-IFN-γ后,将其与免疫佐剂二甲基三十六烷基铵(DDA)混合,皮下免疫C57BL/6小鼠3次,每次间隔2周,末次免疫2周后进行效果评价。采用酶联免疫吸附试验(ELISA)检测血清中IgG、IgG1和IgG2c水平。结果显示,融合蛋白Ag85A-IFN-γ组的IgG水平均高于单独抗原组,且融合蛋白组IgG2c/IgG1比值显著高于对照组,表明融合蛋白能刺激宿主产生更强烈的体液免疫反应,更倾向于激发T辅助细胞1型(Th1型)免疫反应。流式细胞术检测特异性CD4+和CD8+T细胞比例,发现Ag85A-IFN-γ组CD8+/CD4+最高,显示该融合蛋白能显著增强CD8+T细胞增殖。上述结果表明,融合蛋白Ag85A-IFN-γ有望成为有效的新型抗结核融合蛋白亚单位疫苗。

This study aims to evaluate the immunological effects of Mycobacterium tuberculosis protein subunit vaccine candidate Ag85A-interferon y （Ag85A-IFN-y）. Firstly the plasmid pET28a-Ag85A-IFN-y with the open reading frame of mature encoding sequence of Ag85A and IFN-y was constructed, then expressed in Escherichia coil （E. coli） （DE3） BL21 cells. C57BL/6 mice were immunized by Ag85A-IFN-）, three times by subcutaneous injection biweekly. Two weeks after the last immunization, lymphocytes from spleens and sera were separated and detected for assessing the immunological effects. Serum antibodies against Ag85A （IgG, IgG1, and IgG2c） were detected by enzyme-linked immunosorbent assay （ELISA） and spleen lymphocytes subsets were analyzed by flow cytometry. The results demonstrated that the titers of antibodies （IgG, IgG1, and IgG2c） against the fusion protein Ag85A-IFN-y in mice were higher than those in single antigen vaccinated mice and induced a T helper 1 （Thl） cell-mediate immunity response based on the ratio of IgG2c/IgG1. The ratio of CD8＋/CD4＋ in Ag85A-IFN-y vaccinated group was the highest among all groups. The results indicate that the fusion protein Ag85A-IFN-y would be a promising subunit vaccine candidate against tuberculosis.