牛副结核分枝杆菌多组分亚单位疫苗的初步研究

Preliminary study on multicomponent subunit vaccine of bovine Mycobacterium avium subsp. paratuberculosis

为筛选出最佳的预防副结核病的候选疫苗,本研究评价了副结核分枝杆菌(MAP)培养上清、MAP细胞壁和MAP灭活菌体的免疫保护效果。将160只6周龄雌性BALB/c小鼠随机分为8组,每组20只,分别为PBS组、菌影(Ghost)组、弗氏不完全佐剂(IFA)组、上清+Ghost组、上清+IFA组、细胞壁+Ghost组、细胞壁+IFA组和灭活苗组,分别经皮下注射免疫后在不同的时间点采集小鼠尾静脉血,利用间接ELISA检测小鼠血清中抗体效价。三免后以5×10~8cfu/只剂量进行腹腔攻毒。结果显示:细胞壁+IFA组小鼠的抗体水平明显高于其它组(p<0.001),细胞壁+Ghost组次之(p<0.001);除阴性对照组外,各组小鼠IgG1的滴度均高于IgG2a (p<0.001);细胞壁+IFA组和细胞壁+Ghost组的小鼠脾脏淋巴细胞培养上清中IFN-γ、IL-4和IL-10的含量显著高于其它实验组(p<0.001);细胞壁+IFA组IL-2的含量明显高于其它实验组(p<0.001),细胞壁+Ghost组次之(p<0.01);细胞壁+IFA组小鼠的肝脏、脾脏和小肠组织的荷菌量显著少于其它实验组(p<0.01),细胞壁+Ghost组次之(p<0.05),表明MAP细胞壁可以刺激机体产生较高水平的抗体,机体的免疫反应倾向于体液免疫,且细胞壁对攻毒小鼠的保护效果最好。本研究为反刍动物副结核病疫苗的研制提供了实验依据。

In order to screen out the best candidate vaccine against paratuberculosis, the immunoprotective effects of Mycobacterium avium subsp. paratuberculosis （MAP） culture supernatant, MAP cell wall and MAP inactivated bacteria were evaluated in mice by challenge test. In this study, 6-week-old female BALB/c mice were divided into 8 group （20 mice in each group）, and the mice were subcutaneously immunized PBS, Ghost, IFA, Supernatant＋Ghost, Supernatant＋IFA, Cell wall＋Ghost, Cell wall＋IFA, and inactivated vaccine. The blood samples were collected from the mice via tail vein at different time points for the serum antibody detection by ELISA, and the mice were challenged intraperitoneally with 5×108cfu of MAP after the third immunization. The results showed that the antibody level in Cell wall＋IFA group was significantly higher than that of other groups （p〈0.001）, Cell wall＋Ghost group was the second （p〈0.001）. In all groups, except the control, the titer of IgG1 was higher than that of IgG2a （p〈0.001）. The levels of IFN-γ, IL-4, and IL-10 in the spleen lymphocyte culture medium in the Cell wall＋IFA group and Cell wall＋Ghost group were significantly higher than that of other groups （p〈0.001）. The IL-2 level in the Cell wall＋IFA group was significantly higher than that in other groups （p〈0.001）, followed by the Cell wall＋Ghost group （p〈0.01）. The bacteria burden in the liver, spleen and small intestine tissues of mice in the Cell wall＋IFA group were less than that of other groups （p〈0.01）, followed by the Cell wall＋Ghost group （p〈0.05）. The results showed that the Cell wall group was able to stimulate the body to produce higher titer antibodies, and the immune response tends to humoral immunity. Moreover, the Cell wall group offered the highest protection for the mice against the challenge. This study provides experimental evidence for the development of paratuberculosis vaccines for ruminants.