兔多杀性巴氏杆菌铁调节外膜蛋白的小鼠免疫效果分析

Evaluation of the Immunization of Iron-Regulated Outer Membrane Proteins (IROMPs) of Leporid Pasteurella multocida in Mice Model

【目的】本试验以小鼠为动物模型,比较了兔多杀性巴氏杆菌铁调节外膜蛋白(IROMPs)的免疫原性及分析交叉免疫保护效果。【方法】分别以正常培养基和限铁培养基增殖兔多杀性巴氏杆菌C51-12和C51-3,采用Sarcosyl沉淀—超离法提取IROMPs和OMPs,并用SDS-PAGE比较两株之间的差异,同时用正常培养基增殖该菌制备灭活全菌抗原。免疫原性分析,将清洁级小鼠20只随机分成4组,5只/组,以C51-12株的全菌、IROMPs和OMPs作抗原分别免疫,采用间接ELISA检测免疫小鼠的相应抗体动态变化;免疫保护试验,分别采集经4次免疫后小鼠的抗血清,作1﹕10稀释腹腔注射小鼠,每组6只,18h后用C51-12和C51-3分别攻毒,比较上述抗血清所提供的交叉免疫保护力。【结果】前21d,抗体滴度以全菌免疫组抗体滴度较高,随后IROMPs免疫组反超。最终抗体滴度:IROMPs组>OMPs组>全菌组。被动免疫时全菌抗血清和OMPs抗血清能够抵抗C51-12株攻毒,但对C51-3株提供的保护力较差;C51-12株攻毒,IROMPs抗血清组能够全部保护,C51-3株攻毒,5/6获得保护。【结论】IROMPs在小鼠动物模型中具有一定的交叉保护力,为进一步研究交叉保护因子奠定基础。

[Objective] To evaluate the diversity of immunogenicity of IROMPs, OMPs and whole-cell bacterin （WCB） of leporid P. multocida, and evaluate their cross protection in mouse model. [Method] 20 mice were equally assigned to 4 groups at random. They were immunized by IROMPs, OMPs and WCB respectively. The indirect ELISA was used to detect antibody responses. After two months post-vaccinationl, the antisera to these three immunogens and normal serum were collected and diluted with sterile physiological saline （1 ： 10 V/V）. Then 48 mice were equally assigned to 8 groups at random, each group was given cognat antisera by intraperitoneal injection, group a and b, antisera to IROMPs; group c and d, antisera to OMPs; group e and f, antisera to WCB; group g and h, normal serum. Then group a, c, e and g were challenged with P. multocida C51-12 and group b, d, f and h were challenged with C51-3 to observe the passive protection. [Result] The results of active immunity showed that titers of serum antibodies of group WCB were higher than those of group IROMPs and OMPs before 21d. After that, antibody response of group IROMPs climbed rapidly and soon bypassed the WCB group. At last, the titers were： IROMPs group〉WCB group〉OMPs group. The results of passive protection indicated IROMPs-antisera could enhance resistance against P. multocida C51-3 and C51-12, while OMPs-antisera and WCB-antisera weren＇t effective against C51-3. [Conclusion] These results demonstrated that IROMPs could enhance resistance against challenge of heterologous serotype of P.multocida, maybe had characteristics of cross-protection factors （CPFs）.