副结核分枝杆菌免疫原蛋白的筛选及免疫保护效果评价

Screening of Mycobacterium Avium Subsp.Paratuberculosis Immunogenic Proteins and Its Evaluation of Immunological Effect

【背景】副结核病(paratuberculosis,PTB)是由副结核分枝杆菌(Mycobacterium avium subsp.paratuberculosis,MAP)主要引起反刍动物的一种慢性消耗性传染病。患病动物表现为肉芽肿性肠炎和肠道的功能失调、顽固性腹泻、渐进性消瘦、营养不良、贫血、嗜睡甚至死亡。PTB给畜牧业造成巨大的经济损失,并且严重威胁公共卫生安全。由于目前临床上对于PTB的检测和控制手段不够完善,现有的PTB疫苗保护效果不佳,并且干扰牛结核病的诊断,因此亟需研发免疫原性强、保护效果佳的疫苗以用于PTB的防控。【目的】通过筛选MAP免疫原蛋白,并对其免疫保护效果进行评价,为PTB的防控提供数据支持。【方法】根据MAP的p22、map1272c、map3531c、map3783、map3701c以及map3527这6个基因,分别构建5种重组质粒,在获得5种重组蛋白基础上,联合MONTANIDE ISA 61 VG佐剂皮下多点注射免疫小鼠,通过IFN-γELISPOT试验筛选出最佳免疫原;随后将最佳免疫原和已报道的66NC融合蛋白混合,经皮下多点注射免疫小鼠,在二免3周后用1×10^(8)CFU的MAP K-10菌株腹腔感染小鼠。通过IFN-γELISPOT试验,抗体监测和细胞因子检测,分析感染后小鼠的体重、肝脏病理学和组织病理学变化及其荷菌数差异,综合评价候选亚单位疫苗的免疫原性和免疫保护效果。【结果】以MAP p22、map1272c、map3531c、map3783以及map3701c为基础成功构建和表达了5种融合蛋白58F、62F、69F、46F和52F,其中免疫58F后产生的IFN-γ水平最高,是更具潜力的候选免疫原,且以融合蛋白组合66NC+58F诱导持久高滴度的IgG、IgM、IgG1和IgG2a水平,诱导特异性的IFN-γ、TNF-α和IL-17A的释放;在保护效果评价中,融合蛋白组合66NC+58F抵抗MAP感染造成的体重下降,显著减轻肝脏的病理损伤,降低MAP在肝脏中的定植。【结论】融合蛋白组合66NC+58F诱导小鼠产生Th1和Th17型免疫反应,对MAP感染有着一定免疫保护作用,是PTB重要的候选亚单位疫苗。

【Background】Paratuberculosis(PTB)is a chronic,wasting infectious disease caused by Mycobacterium avium subsp.paratuberculosis(MAP)in ruminants.PTB causes huge economic losses to the livestock industry and poses a serious threat to public health safety.Since the current clinical methods for the detection and control of PTB are inadequate,and the PTB vaccine used is ineffective and interferes with the diagnosis of bovine tuberculosis,there is a need for developing a vaccine with strong immunogenicity,good safety,and excellent protection for the prevention and control of PTB.【Objective】The immunogenic protein of MAP was screened and its immunoprotective effect was evaluated,so as to provide the data support for the prevention and control of PTB.【Method】Five recombinant plasmids were constructed based on six genes of MAP:p22,map1272c,map3531c,map3783,map3701c,and map3527.The five recombinant proteins were combined with MONTANIDE ISA 61 VG adjuvant to immunize mouse by subcutaneous injection,and the best immunogen was screened by IFN-γELISPOT assay.The best immunogen was then mixed with the reported 66NC fusion protein.Mouse were immunized by subcutaneous multi-point injection.At 3 weeks after the second immunization,mice were immunized with 1×10^(8)CFU of the MAP K-10 strain intraperitoneally.The immunogenicity and immunoprotective effect of the candidate subunit vaccine were comprehensively evaluated by IFN-γELISPOT assay,monitoring antibody titers and serum cytokines,as well as detecting weight changes,liver pathological and histopathological observations and charge count differences of infected mouse.【Result】Five recombinant proteins,such as 58F,62F,69F,46F,and 52F,were expressed based on the genes p22,map1272c,map3531c,map3783 and map3701c.58F produced the highest level of IFN-γafter immunization and was the most promising candidate immunogen.The fusion protein combination 66NC+58F induced persistent high titers of IgG,IgM,IgG1 and IgG2a,and also induced specific release of IFN-γ,TNF-α,and IL-17A.In the evaluation of protective effects,the fusion protein combination 66NC+58F resisted the weight loss caused by MAP infection,significantly reduced pathological damage in the liver,and decreased MAP colonization in the liver.【Conclusion】The fusion protein combination 66NC+58F induced Th1 and Th17-type immune responses in mouse,provided immune protection against MAP infection and was an important candidate subunit vaccine for PTB.