Objectives: With the decreased protective effect of Bacille Calmette-Guérin (BCG) vaccine, widespread drug-resistant strains of tuberculosis as well as the lack of effective vaccines, global morbidity and mortality of tuberculosis remains high. The purpose of this study is to evaluate the potential of Mycobacterium tuberculosis antigen Rv2654 as a candidate vaccine against tuberculosis, and to verify the characteristics of cellular and humoral immune responses in mice induced by this protein.
Methods: Isopropyl-beta-D-thiogalactoside (IPTG) was added to induce the expression of Rv2654 recombinant protein in the logarithmic growth stage of bacteria. The recombinant protein was purified by affinity chromatography and identified by Western blotting. The immunoreactivity of Rv2654 recombinant protein with human sera was detected by ELISA. After immunization with Rv2654 recombinant protein, the levels of Th1/Th2 cytokines in peripheral blood of mice was measured using cytokine magnetic bead arrays, and the T and B lymphocyte subsets in spleen of mice was analyzed by flow cytometry.
Results: The recombinant protein Rv2654 was successfully induced and expressed. The ELISA data showed that the recombinant protein Rv2654 was responsive to the serum of BCG-inoculated patients or active pulmonary tuberculosis patients. In immunized mice with recombinant protein Rv2654, the expression of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and other cytokines in peripheral blood was elevated (all P<0.05). Flow cytometry analysis showed that the recombinant protein significantly stimulated the differentiation of CD4+T cells and CD8+T cells into effector T cells, and this effect was more obvious when combined with BCG. The recombinant protein Rv2654 also stimulated the activation and proliferation of B cells and their differentiation into memory cells. However, less plasma cells were produced.
Conclusions: Rv2654 protein could induce the cell immune responses and it has good binding ability with serum of BCG-inoculated patients and active pulmonary tuberculosis patients, suggesting that it may serve as a good candidate antigen for tuberculosis immunological prophylaxis and diagnostic vaccines.
目的: 随着卡介苗的保护效应日趋减弱，以及结核分枝杆菌耐药株的广泛存在，全球尚缺乏控制结核病的有效疫苗，全球结核病发病率和病死率仍然较高。本研究旨在分析结核分枝杆菌抗原Rv2654重组蛋白在诱导小鼠细胞免疫和体液免疫应答中的作用，评价其作为抗结核病候选疫苗的潜力。方法: 在细菌的对数生长期加入诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactoside，IPTG)诱导细菌表达Rv2654重组蛋白，用亲和层析纯化，蛋白质印迹法鉴定Rv2654重组蛋白。采用ELISA方法检测不同人群血清与Rv2654重组蛋白的免疫反应性。以Rv2654重组蛋白免疫昆明小鼠，采用细胞因子磁珠阵列法检测小鼠外周血Th1/Th2类细胞因子水平，并以流式细胞术分析小鼠脾T、B淋巴细胞亚群分布。结果: 成功表达Rv2654重组蛋白，以Rv2654重组蛋白作为抗原的ELISA结果显示其与卡介苗接种者或活动期结核患者血清均有反应性。Rv2654重组蛋白免疫小鼠可促进小鼠外周血IFN-γ、TNF-α、IL-2、IL-4、IL-6和IL-10等多种细胞因子表达(均P<0.05)，流式细胞术结果显示Rv2654重组蛋白可显著刺激CD4+T细胞和CD8+T细胞分化为效应T细胞，这种作用在联合卡介苗应用时更为明显，Rv2654重组蛋白也能刺激小鼠B细胞活化增殖并向记忆细胞分化。结论: Rv2654重组蛋白可诱导小鼠的细胞免疫应答，而且与卡介苗接种者和活动性肺结核患者血清有较好的结合能力，有望作为结核病免疫学预防和诊断类疫苗的候选抗原。.
Keywords: Mycobacterium tuberculosis; Rv2654 recombinant protein; cellular immunity; humoral immunity; tuberculosis vaccine.