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【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。
【Objective】 Soluble Mycobacterium tuberculosis Ag85A protein was expressed and its immunogenicity was evaluated. 【Method】 Soluble Ag85A protein was expressed in E. coli with cold shock expression plasmid and chaperone plasmid, and purified and identified. The immunogenicity of Ag85A protein in C57BL / 6 mouse model was evaluated by inducing body-specific humoral Immune response and cellular immune response levels were analyzed. 【Result】 The results showed that the soluble Ag85A protein was detected in the supernatant after induced by recombinant bacteria. The purity of Ag85A protein was over 90% purified by affinity chromatography. Western blot showed that Ag85A protein had good immunoreactivity. Ag85A protein immunized mice, the serum can detect high levels of Ig G antibody titers, which Ig G2b levels higher than Ig G1. Through specific peptides, protein-stimulated spleen and inguinal lymph node cells can secrete high levels of Th1-type cytokines such as IFN-γ, TNF-α and so on. 【Conclusion】 Soluble expression of Ag85A protein was achieved. Immunological evaluation of Ag85A protein showed that Ag85A protein induced strong humoral and Th1-type cellular immune responses, which laid an important foundation for its further immunological function.