结核杆菌PEPCK表达及保护性免疫反应效果  

Expression of phosphoenolpyruvate carboxykinase of Mycobacterium bovis BCG and relative immune responses in mice

作　　者：柏雪莲[1] 魏庆宽[2] 李瑾[2] 李桂萍[2] 韩广东[2] 崔勇[2] 刘克义 

机构地区：[1]滨州医学院病原生物学教研室,山东滨州256603 [2]山东省寄生虫病防治研究所 [3]美国康乃尔大学

出　　处：《中国公共卫生》2009年第5期513-515,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(30340073)

摘　　要：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 express the recombinant protein of phosphoenolpyruvate carboxykinase （PEPCK） of Mycobacterium bovis and research the protective immune responses induced by the protein. Methods The recombinant plasmid of pHF/pckA which included pckA gene of Mycobacterium bovis was transformed into Escherichia coli HB101 （E. coli HB101 ）. The recombinant protein of PEPCK was expressed and identified by SDS-PAGE and Western blot. Sixty BALB/c mice were divided into two groups. Each mouse in experimental group was injected with emulsion containing 10 μg PEPCK antigen（Ag） in PBS and equal volumes of incomplete Freund＇s adjuvant（Difco） through the peritoneal. In control group, each mouse was only injectedwith equal volumes of incomplete Freunds adjuvant. All mice were immunized for three times at an interval of two weeks. Spleens and blood were taken for the detection of CD4^＋ ,CD8^＋ T cells and cytokines 2 weeks after the final immunization. Results The recombinant PEPCK was expressed and identified by Western blot. The spleen of mouse in PEPCK immunized group was much bigger than that of control group. The number of CD4^＋ T cells and the ratio of CD4^＋/CD8^＋ in the PEPCK immunized group were significantly higher than that of control group （ P 〈 0. 01 ）. The amount of interferon-gama,interleukin-12 and tumor necrosis factor-alpha in PEPCK immunized mice was higher than that of control group（ P 〈 0. 05 ）. Conclusion PEPCK can induce strong cellular and humoral immune responses and may be a good candidate for TB vaccine.

关 键 词：结核杆菌 磷酸烯醇型丙酮酸羧激酶 免疫保护 

分 类 号：R378.911[医药卫生—病原生物学]