猪传染性胸膜肺炎重组亚单位疫苗在小鼠体内的免疫机制及其保护效力

Protective Efficacy and Immunologic Mechanism of the Recombinant Subunit Vaccine Against Actinobacillus pleuropneumoniae in Mice

【目的】欲构建具有较好交叉免疫保护效果的多组分重组亚单位疫苗,从而为猪传染性胸膜肺炎新型疫苗的研制提供参考。【方法】利用分子克隆和重组表达技术获得了猪传染性胸膜肺炎放线杆菌6种主要毒力因子rApxⅠ、rApxⅡ、rApxⅢ、rApxⅣ、rApfa和rOMP。并设立以灭活苗(5×108cfu)为试验Ⅰ组,以重组蛋白rApxⅠ、rApxⅡ、rApxⅢ和rOMP为试验Ⅱ组,以rApxⅠ、rApxⅡ、rApxⅢ、rApxⅣ、rApfa和rOMP为试验Ⅲ组,以PBS为空白对照组。分3次免疫BALB/c小鼠,每次间隔2周,第3次免疫后的第7天以APP1型(5×109cfu)和APP2型(5×1010cfu)进行攻毒保护试验。【结果】试验Ⅱ组的4种抗体水平(ApxⅠ、ApxⅡ、ApxⅢ和OMP)和脾淋巴细胞诱生IL-2水平显著高于其它3组(P<0.05),脾淋巴细胞增殖水平也一直高于其它3组(P>0.05)。且试验Ⅱ组对APP1型保护作用(9/10)明显高于试验I组(6/10)、试验Ⅲ组(5/10)和对照组(0/10),而对APP2型保护作用(无肺脏损伤)也明显优于其它3组(典型肺部损伤),显示出细胞免疫和体液免疫水平与免疫攻毒保护之间存在着正相关性。【结论】试验Ⅱ组通过激活体液免疫和细胞免疫,对不同血清型APP的攻击提供了很好的交叉保护作用。

[ Objective ] To construct a multi-component recombinant subunit vaccine that could provide favorable cross-protection, which provides data for the research of novel porcine contagious pleuropneumonia（PCP） vaccines. [Method] Six major virulence factors of Actinobacillus pleuropneuminiae（APP）, rApxⅠ, rApxⅡ, rApxⅢ, rApxⅣ, rOMP and rApfa were recombinanfly expressed. Using the inactivated vaccine of APP serotype 1 （5×10^8cfu） as group Ⅰ, the recombinants of rApxⅠ, rApxⅡ, rApxⅢ and rOMP as group Ⅱ, that of rApxⅠ, rApxⅡ, rApxⅢ, rApxⅢ, rApfa and rOMP as group Ⅲ, and PBS as the control group, the tests of immuno-protection against challenge were performed respectively, in which BALB/c mice were immunized three times at two week intervals and challenged with serotype 1（5×10^9cfu） and serotype 2 （5×10^10cfu）intranasally at the seventh day after the third immunization. [Result] The results suggested that group Ⅱ had a higher antibody level to four antigens and IL-2 production than others （P〈0.05）. It had a slightly higher lymphocytes proliferation than others （P〉0.05）. The protective efficacy against serotype lof group Ⅱ （9110） was obviously better than group Ⅰ（6/10）, group Ⅲ（5/10） and the control group（0/10）, and the protective efficacy against serotype 2 of that （no lung lesion） was obviously better than others（typical lung lesion）., which demonstrated that the antibody level had a positive correlation to protective efficacy. [Conclusion] The recombinant subunit vaccine containing ApxⅠ,ApxⅡ, ApxⅢ and OMP provided significantly crossing protection by simutaneously stimulating humoral immunity and cell immunity.