结核杆菌磷酸烯醇型丙酮酸羧基酶诱导小鼠细胞和体液免疫应答

目的检测结核杆菌磷酸烯醇型丙酮酸羧激酶(phosphoenolpyruvate carboxykinase,PEPCK)对BALB/c小鼠的免疫保护作用。方法选清洁级BALB/c小鼠60只,随机分为两组,实验组和对照组。实验组每只小鼠用表达的PEPCK融合蛋白10μg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2w免疫1次,共免疫3次。末次免疫2w后,分别取小鼠脾脏、血清,流式细胞仪检测CD4+和CD8+T细胞,MTT法检测淋巴细胞对刺激的应答水平,ELISA检测血清各种细胞因子及抗PEPCK抗体。结果实验组小鼠的脾脏明显大于对照组小鼠的脾脏,且粘连严重,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);且淋巴细胞的应答能力明显强于对照组小鼠;实验组小鼠血清中IFN-γ、IL-12和TNF-α明显高于对照组,血清抗体滴度随免疫次数逐步增高。结论结核杆菌PEPCK能够有效的刺激机体产生细胞免疫反应和体液免疫反应,尤以细胞免疫反应为主,是很好的抗结核候选疫苗分子之一。

Celluar and humoral immune responses induced by phosphoenolpyruvate carboxykinase of Mycobacterium bovis BCG

To research the immune protection induced by phosphoenolpyruvate carboxykinase(PEPCK) of Mycobacterium tuberculosis to mice,sixty BALB/c mice were divided into two groups.Each mouse in experimental group was injected with Emulsions comprising 10μg PEPCK antigen(Ag) in PBS and equal volumes of incomplete Freund's adjuvant(Difco).In control group,each mouse was only injected with equal volumes of incomplete Freund's adjuvant.All mice were immunized for three times with an interval of two weeks.Spleens and blood were taken 2 weeks after the final immunization.The amount of CD4+ and CD8+ T cells was assayed by flow cytometry.The response capability of lymphocytes was detected by MTT.At the same time cytokines such as IFN-γ,IL-12 and TNF-αwere detected by ELISA.The spleen of mouse in experiment group was much bigger than that of control group.The number of CD4+ T cells and the ratio of CD4+/CD8+ from experiment group were significantly higher than that of control group(P<0.01).And the lymphocytes' responsion capability of mice in experiment group was stronger than that of control group.The amount of IFN-γ,IL-12 and TNF-α in PEPCK immunized mice was also higher than that of adjuvant group(P<0.05).The titer of antibody in serum of PEPCK immunized mice got higher along with the immune times.The results showed that PEPCK could induce strong cellular and humoral immune responses,especially cellular responses and may be a good candidate for TB vaccine.