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目的研究结核杆菌磷酸烯醇型丙酮酸羧激酶(PEPCK)的表达,并观察此融合蛋白使机体产生的保护性免疫反应。方法将含有结核杆菌磷酸烯醇型丙酮酸羧激酶(pckA)基因的重组质粒转化大肠埃希菌HB101.,表达出融合蛋白PEPCK,并进行蛋白质印迹(Western blot)分析;选BALB/c小鼠60只,随机分为实验组和对照组。实验组每只小鼠用表达的PEPCK10μg加弗氏不完全佐剂进行腹腔免疫注射,对照组小鼠仅用弗氏不完全佐剂注射。每隔2周免疫1次,共免疫3次。末次免疫2周后,分别取小鼠脾脏、血清,检测CD4+T细胞和CD8+、T细胞及各细胞因子。结果成功表达出融合蛋白PEPCK,并能够与小鼠抗卡介苗(BCG)血清反应;实验组小鼠的脾脏明显增大,粘连严重;CD4+T细胞增殖明显(73.5±3.69),CD4+/CD8+比值显著升高(5.1±0.98)(P<0.01);血清中γ干扰素(IFN-γ)、白介素12(IL-12)和α肿瘤坏死因子(TNF-α)均有不同程度的升高。结论PEPCK能够诱发小鼠产生细胞免疫和体液免疫反应,是很好的抗结核疫苗候选分子之一。
Objective To study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in Mycobacterium tuberculosis and to observe the protective immune response induced by this fusion protein. Methods The recombinant plasmid containing mycobacterium tuberculosis phosphoenolpyruvate carboxykinase (pckA) gene was transformed into Escherichia coli HB101. The fusion protein PEPCK was expressed and analyzed by Western blot. BALB / c mice 60 were randomly divided into experimental group and control group. Each mouse in the experimental group was immunized intraperitoneally with PEPCK 10 μg of incomplete Freund’s adjuvant and the control mice were injected with incomplete Freund’s adjuvant alone. Immunization once every 2 weeks, a total of 3 immunizations. Two weeks after the last immunization, the spleen and serum of mice were taken respectively to detect CD4 + T cells, CD8 + T cells and various cytokines. Results The fusion protein PEPCK was successfully expressed and could react with the anti-BCG serum of mice. The spleen of the mice in the experimental group was significantly increased and the adhesions were severe. The proliferation of CD4 + T cells was significantly (73.5 ± 3.69) and CD4 + / CD8 + (5.1 ± 0.98) (P <0.01). The levels of IFN-γ, IL-12 and TNF-α in serum increased to some extent. Conclusions PEPCK can induce cellular and humoral immune responses in mice and is one of the best candidates for antituberculosis vaccine.