结核分枝杆菌Rv2628蛋白的免疫生物学特性

Immunological characteristics of Mycobacterium tuberculosis antigen Rv2628

摘要:

Rv2628蛋白是结核分枝杆菌Mycobacterium tuberculosis (M. tb) DosR调控的潜伏感染相关抗原。本研究对Rv2628蛋白进行了原核表达和纯化，并以巨噬细胞系和小鼠为研究模型，对其免疫生物学特性进行了分析。SDS-PAGE及Western blotting鉴定结果表明，Rv2628-His融合蛋白以包涵体形式表达，能与兔抗H37Rv多抗血清发生特异性反应，具有较好的免疫反应性。与巨噬细胞系RAW264.7的互作实验结果表明，在1–12 h内Rv2628蛋白能诱导前炎性因子IL-6的上调表达。将纯化的Rv2628融合蛋白皮下免疫BALB/c小鼠，夹心ELISA的测定结果表明，Rv2628蛋白免疫组诱导产生的特异性IFN-γ水平显著高于IL-4的水平 (P＜0.000 1)，呈现Th1型细胞免疫应答趋势；以Rv262811-30多肽作为包被抗原，通过间接ELISA测定的血清抗体效价能达到11 600，表明Rv2628也能诱导体液免疫应答。总之，Rv2628能促进巨噬细胞炎症反应的发生，激发小鼠产生强烈的Th1型细胞免疫应答和较好的体液免疫应答，具有作为亚单位疫苗的潜力，为M. tb与宿主之间的相互作用奠定了一定的理论基础。

Abstract:

Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1–12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-γ and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-γ was significantly higher than that of IL-4 (P<0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv262811-30 peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.