支气管败血波氏杆菌外膜蛋白的提取及其免疫效果的检测

Extraction and Detect on Immunization of Outer Membrane Protein of Bordetella bronchiseptica

摘要:

本研究以小鼠为实验动物模型, 研究支气管败血波氏杆菌的外膜蛋白(OMP)和其中的有效保护抗原成分(OMP68)的免疫原性及免疫保护作用。本研究利用改进的Wooldridge的方法提取了支气管败血波氏杆菌P11和P13的OMP, 利用SDS-PAGE比较了2株之间差异, 采用Western-blotting进行了分析并确定了P13菌株OMP(P13-OMP)中的有效保护性抗原成分, 采用电洗脱方法获的分子量为68 kD的P13-OMP有效保护抗原成分(OMP68), 然后制备了油乳剂P13的全菌、P13-OMP和OMP68免疫抗原。抗体动态变化：将清洁级小鼠70 只随机分成7 组, 10 只/组, 用油乳剂P13的全菌和不同剂量的P13-OMP和OMP68免疫抗原分别免疫, 采用间接 ELISA 检测免疫小鼠的相应抗体水平并分析抗体动态变化; 主动免疫保护试验：将清洁级小鼠80 只随机分成8 组, 10 只/组, 分别采用制备的油乳剂P13-OMP(OMP25 μg/只)、OMP68(25 μg/只)和P13菌体免疫抗原在免疫10 d后, 分别用100 LD50的P11和P13腹腔攻毒, 然后统计保护率并分析免疫原所提供的免疫保护力。免疫P13-OMP和OMP68油乳剂抗原后, 7 d时, 抗体水平开始逐渐上升, 42 d时, 抗体水平分别达到215.3和214.8, 然后逐渐下降, 70 d时, 抗体水平分别达到29.1和210.2。主动免疫时, P11和P13攻毒的P13-OMP免疫组分别均得到9/10和9/10的保护, OMP68免疫组分别均得到9/10和10/10的保护, 而P13全菌免疫组分别得到4/10和6/10保护, 对照组全部死亡。P13-OMP和OMP68抗原均具有良好的免疫原性和免疫保护作用, 为支气管败血波氏杆菌OMP亚单位疫苗的研制奠定了良好的理论基础。

关键词:支气管败血波氏杆菌, 小鼠, 外膜蛋白, 免疫原性

Abstract:

In order to evaluate the immunogenicity and their protection of outer membrane protein of Bordetella bronchiseptica and effective antibody (OMP68, we established a mice model. The research improved on the method of Wooldridge in order to extract OMP of Bordetella bronchiseptica (P13, P11), and the differences of OMP were analyzed by SDS-PAGE. The P13-OMP was analyzed with Western blotting and OMP68 was gained by the way of electrophoretic elution. Then we maked the effective antibodies of P13-OMP. Then P13-OMP, OMP68 and whole-cell bacterin (WCB)immunizing antigen was prepared 70 mice were equally assigned to 7 groups at random. They were immunized by P13-OMP, OMP68 and P13-WCB of different dose respectively. The indirect ELISA was used to detect antibody responses. 80 mice were equally assigned to 8 groups at random. Group a and b were immunized by P13-OMP (25 μg) respectively. Groups c and d were immunized by OMP68(25 μg); Groups e and f were immunized by WCB(P13). Ten days after the immunizations, group a, c, e and f were challenged with P13. The other groups were challenged with P11. Then we registered the rate of protective potency and evaluated protection given by P13-OMP, OMP68 and P13-WCB. Results showed that at 42th day, the titer of serum reach the top: P13-OMP(215.3)> OMP68(214.8). Then, the titer of serum started the slow-moving descent At 70th day, the titer of serum reach the OMP68 (210.2)> P13-OMP (29.1). P13-OMP and OMP68 group challenged with P13 and P11 can be efectivly protected; P13-WCB group challenged with P13 and P11 can not be efectivly protected; the control group were died out. The P13-OMP and OMP68 of Bordetella bronchiseptica has good immunogenicity and protection, so the results of this study lay good theoretical foundation for OMP subunit vaccine.

Key words:Bordetella bronchiseptica, Mice, Immunogenicity, OMP