摘要
猪胸膜肺炎放线杆菌是引起猪胸膜肺炎的主要病原,该病原引起的猪传染性胸膜肺炎是一种具有高度传染性的呼吸道病,世界各国均有发生,给世界范围内养猪产业造成了巨大的经济损失。该菌的15个血清型均能致病,防制难度较大。我国流行的优势血清型是1、2、3、7型,本实验室已完成了对血清3型JL03株的全基因组测序工作。基于以上基础,本研究旨在应用反向疫苗学技术筛选猪胸膜肺炎放线杆菌保守的免疫原性蛋白,结合新型O/W佐剂MF59研究具有交叉保护效果的亚单位疫苗。取得的主要研究结果如下：
1.猪胸膜肺炎放线杆菌免疫原性蛋白的筛选与鉴定
应用免疫蛋白质组技术对猪胸膜肺炎放线杆菌血清3型JL03株进行了研究,用康复血清从其外膜蛋白和分泌蛋白中筛选到33个具有免疫反应性的蛋白,分泌蛋白16种,外膜蛋白17种,其中有三种蛋白MomP1、MomP2、EF-Tu在分泌蛋白和外膜蛋白中同时被鉴定。在鉴定的30个免疫原性蛋白中有6个蛋白是已知的抗原蛋白,24个蛋白是猪胸膜肺炎放线杆菌的新型免疫原性蛋白。新型免疫原性蛋白中已经在其它病原中表现出免疫原性的蛋白有D15/OmpD、LppB、FrdA、MDH、FepA、FrpB、TufB、PotD、GapA、ZnuA、TIG、DegP、TufB、PsaA、FkpA、PTA;有一部分蛋白在本研究究中被首次鉴定为免疫原性蛋白,但其功能在一些细菌中已经得到证实,这些蛋白是CbiK、IlvG、FepB、AfuC、FatB、GGBP、CysG、Ttg2D。
2.免疫原性蛋白亚细胞定位及小鼠免疫保护效果评价
对筛选到的蛋白进行生物信息学分析后,我们选取了通过间接免疫荧光试验证实分布于APP表面的蛋白OmpD、LppB、FepA作为研究对象,对其基因进行体外的克隆和表达,纯化后的蛋白通过小鼠免疫攻毒试验进行评价,结果表明,免疫小鼠在APP1型菌株攻毒后,OmpD、LppB对小鼠的保护率均为70%,FepA对小鼠的保护率仅为40%。
3.亚单位疫苗的研究ApxⅠ、ApxⅡ、ApxⅢ是APP重要的毒素及抗原蛋白,应用本实验室构建保存的表达质粒表达这三种毒素蛋白的抗原部份ApxⅠA、ApxⅡA、ApxⅢA,将纯化的rApxⅠA、rApxⅡA、rApxⅢA与对小鼠免疫保护率较高的rOmpD、rLppB组合,与新型佐剂MF59混合制备成抗原含量不同的两种亚单位疫苗,疫苗Ⅰ(各抗原组份50μg/ml)和疫苗Ⅱ(各抗原组份75μg/ml)。
用小鼠模型对疫苗Ⅰ、Ⅱ进行安全性和免疫效果评价。采用2倍免疫剂量腹腔接种小鼠后,观察14天,小鼠采食、精神状态无异常,表明疫苗Ⅰ、Ⅱ对小鼠是安全的。之后用制备好的疫苗Ⅰ、Ⅱ和疫苗Ⅲ(商品化的油佐剂灭活疫苗含血清1、2、7型菌株)对小鼠先后进行两次免疫,于不时间对亚单位疫苗各组份抗原的特异性抗体水平进行检测,并对IgG抗体亚类IgG1和IgG2a进行检测,在二免后10天使用我国优势血清型APP 1、2、3、7型标准菌株5LD50剂量对免疫组和对照组小鼠进行攻毒。结果显示,使用疫苗Ⅰ、Ⅱ免疫的小鼠各抗原组份抗体水平均极显著的高于灭活疫苗,这主要是由于灭活疫苗中不含有ApxⅠA、ApxⅡA、ApxⅢA这三种毒素蛋白,而OmpD和LppB蛋白在整个菌体蛋白中所占的比例不高所致；在整个免疫过程中,疫苗Ⅰ、Ⅱ免疫的小鼠均能产生较高的IgG1、IgG2a抗体水平,表明疫苗Ⅰ、Ⅱ能诱导免疫小鼠产生特异的体液免疫反应和细胞免疫反应,但IgG1的抗体水平显著的高于IgG2a的水平,说明疫苗Ⅰ、Ⅱ免疫小鼠后所诱导的免疫反应偏向于以Th2型免疫反应为主；疫苗Ⅰ、Ⅱ组在1、2、3、7型菌株5LD50剂量攻毒后,免疫小鼠肺脏没有明显的解剖学病理变化,仅在1型菌株攻毒组观察到有轻微的组织学病理变化,疫苗Ⅲ组小鼠用1、2,、7型菌株攻毒后没有明显的解剖病变,也无明显的组织病理学变化；用3型菌株进行攻毒后,疫苗Ⅲ组的小鼠出现了较为严重的解剖学病理变化,出血坏死明显,相应的组织病理学变化也较明显,肺泡间质增厚,大量炎性胞细浸润,肺泡充血、出血。疫苗Ⅰ、Ⅱ组对各血清型攻毒保护率比较接近,在70%-90%之间,大多为80%；疫苗Ⅲ组对1、2、7型菌株的攻毒保护率均达到了100%,但对3型菌株的攻毒保护率仅40%。
此外,在疫苗Ⅲ组免疫小鼠的肝脏表面覆盖有白色的油状物,可能是灭活疫苗中含有不能被机体吸收的油佐剂造成的。而在使用MF59佐剂的疫苗Ⅰ、Ⅱ组均没有观察到这种现象,推测本研究中使用的MF59水包油佐剂具有易被降解吸收的优点。
上述研究结果表明,本研究中的亚单位疫苗对小鼠是安全的,与灭活苗相比,亚单位疫苗接种时应激小,具有较好的交叉保护效果,且使用的佐剂容易被降解吸收,有利于动物福利和生产安全的动物食品。因此本研究的亚单位疫苗具有较好的开发前景,但对猪的免疫保护效果还有待于进一步研究。
Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease and result in severe losses in the swine industry worldwide. The predominant serotypes in China were serotype 1,2,3 and 7 and the genome sequence of serotype 3 strain JL03 have been determined by our lab. Based on these findings, Current study focused on screening conserved antigens from Actinobacillus pleuropneumoniae by reverse vaccinology, and developed subunit vaccines for cross protection in combination with O/W adjuvant MF59.The obtained findings were as follows:
1. Identification of immunogenic proteins from Actinobacillus pleuropneumoniae
In this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 with convalescent sera for the identification of novel immunogenic proteins for A. pleuropneumoniae. A total of 33 immunogenic proteins were identified from outer membrane (17 proteins) and extracellular proteins(16 proteins) of JL03 serotype 3, of which three proteins, MomP1, MomP2, EF-Tu, were identified simultaneously from OMPs and ECPs. Of the 30 identified immunogenic proteins,6 were known antigens and 24 were novel immunogenic proteins. In novel immunogenic proteins,16 proteins have already been shown immunogenic in certain pathogenic bacteria, D15/OmpD, LppB, FrdA, MDH, FepA, FrpB, TufB, PotD, GapA, ZnuA, TIG, DegP, TufB, PsaA, FkpA and PTA, and 8proteins, CbiK, IlvG, FepB, AfuC, FatB, GGBP, CysG and Ttg2D, are reported to be immunogenic proteins for the first time in this study whose functions have been biologically demonstrated in some bacteria.
2. Subcellular localization of immunogenic proteins and protective efficacy in mice
Genes of ompD, lppB, and fepA were cloned and expression in E. coli BL21, proteins were purified and analysed by Western blot, and they were demonstrated to be localized on surface of cell by indirect immunofluorescence. Immunogenecity of OmpD, LppB and FepA were evaluated in mice model, and results showed 70% protective efficacy from OmpD and LppB, but only 40% from FepA.
3. Study on subunit vaccine
Purified rApxⅠA、rApxⅡA、rApxⅢA were in combination with rOmpD and rLppB for subunit vaccine preparation, and MF59 adjuvant were equally mixed (V/V) with prepared antigens. Two subunit vaccines were prepared, Vaccine I contained 50μg/ml of rApxⅠA、rApxⅡA、rApxⅢA, rOmpD and rLppB respectively, and vaccineⅡ75μg/ml. VaccineⅠ, vaccineⅡand VaccineⅢ(inavtivated vaccine, containing APP 1,2,7 serotype strains) were inoculated mice. Antibody titer of rApxⅠA、rApxⅡA、rApxⅢA, rOmpD and rLppB in sera from vaccineⅠandⅡgroups showed significantly higher than sera from control and vaccineⅢgroup. Mice were challenged with 5LD50 serotype 1,2,3,7 strains, survival mice of vaccineⅠ,Ⅱgroups had not shown obviously pathologic and histological changes in lungs, and slight histological changes in lungs after challenge with APP serotype 1. Mice of vaccineⅢgroup had not shown obviously pathologic and histological changes in lungs after challenge with APP serotype 1,2,7, but had shown obvious pathologic and histological changes in lungs after challenge with APP serotype 3. VaccineⅠandⅡgroups showed 70%-90% protective efficacy against challenge with APP serotype,1,2,3,7 strains. However, vaccineⅢgroup showed 100% protective efficacy against challenge with APP serotype 1,2,7 strains, but 40% protective efficacy against serotype 3. Data in the present study demonstrated that subunit vaccines induced cross-protection in comparision with vaccineⅢ(inactivated vaccine), and MF59, an O/W adjuvant, was degradable in vivo. Subunit vaccines in this study are epcected to be a novel vaccine for APP, and protective efficacy in pigs needs investigation in further study.
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