【摘要】 副猪嗜血杆菌(Haemophilus parasuis)是一种常见的猪(Susscrofa domestica)致病菌,其细胞致死性膨胀毒素(cytolethal distending toxin,CDT)被认为是一种重要的毒力因子,为进一步明确CDT的一些生物学及免疫学特性,本研究经体外组装获得了高纯度的CDT三聚体完整毒素,研究三聚体蛋白对Marc145及仔猪外周血淋巴细胞(peripheral blood lymphocyte,PBLC)的毒性作用,明确CDT三个亚基的特异性抗体对其细胞毒性的阻断作用。原核表达的3个CDT亚基经过共折叠形成了稳定的三聚体蛋白,能诱导Marc145细胞产生典型的细胞病变。Cdt B及Cdt C的兔抗血清对CDT完整毒素具有中和作用,且Cdt C的兔抗血清可以更有效地阻断CDT的细胞毒性作用,最大中和效价为1∶16,而Cdt A亚基的兔抗血清无中和作用。CDT可以抑制丝裂原活化的仔猪PBLC增殖作用。致病菌株及非致病菌株分泌蛋白中CDT的滴度一致,均为1∶512。本研究结果表明,副猪嗜血杆菌CDT具有明显的免疫抑制作用,可能对于该菌在宿主体内定殖以及引起宿主系统感染至关重要,但并非造成不同菌株致病力显著差异的原因。本研究为揭示副猪嗜血杆菌的致病机理提供了重要信息。更多还原

【Abstract】 Haemophilus parasuis is a common bacterial agent of porcine(Susscrofa domestica), and the infection of this pathogen is characterized by fibrinous to fibrinopurulent polyserositis and polyarthritis.Cytolethal distending toxin(CDT) is proposed as an important virulent factor of H. parasuis, but some biological and immunological characteristics of CDT are still to be studied. Highly purified CDT holotoxin was prepared through co-refolding of 3 subunits by dialysis at 4 ℃ into a native buffer and purification by NiNTA affinity chromatography and gel- filtration chromatography. Cytotoxicity of CDT on both Marc145 and porcine peripheral blood lymphocyte(PBLC) was studied. Moreover, neutralization effect of specific antibody of CDT subunits against its cytotoxicity was measured. It showed that stable ternary complex were formed following co-refolding with 3 recombinant CDT subunits, and the formation of triplex CDT was confirmed by immunoprecipitation assay with purified Ig G against Cdt C subunit. The CDT holotoxin could induce cell distention by 2~4 fold of the original size and nuclear enlargement on Marc145, while any one or 2 of the 3subunits could not induce typical cytotoxicity. Both Cdt B and Cdt C anti- serum could inhibit cytotoxicity of the assembled CDT holotoxin, and Cdt C anti serum showed the best neutralization effect with the titer of 1∶16, while no neutralization effect of Cdt A anti- serum was observed. The unassembled recombinant subunits mixture was significantly more susceptible to the neutralization effect of both Cdt B and Cdt C anti-serum than the assembled CDT holotoxin(P<0.05). Furthermore, H. parasuis CDT blocked proliferation of mitogen activated PBLC, and the proliferation of PBLC was significantly suppressed when applied 20 or 10 μL holotoxin to Con A or phorbol- My- ryslate- acetate(PMA) activated cells, respectively(P<0.05). When cultured in vitro, virulent and non- virulent H. parasuis secreted proteins showed the same cytotoxicity effect with the titer of 1∶512; The result of immunoprecipitation assay with purified Ig G against Cdt C subunit showed that CDT in the secreted proteins was present as ternary complex. Hence, H. parasuis CDT recombinant subunits could form holotoxin in vitro, and the assembled holotoxin exhibited effective bioactivity; Humoral immune responses against Cdt B and Cdt C could completely block the cytotoxicity caused by the holotoxin; Holotoxin showed significant proliferation suppression effect on mitogen activated PBLC cells; H. parasuis secreted proteins of 2 strains with great variation in virulence showed the same cytotoxicity,and CDT in the secreted proteins was presented as triplex holotoxin. H. parasuis CDT exhibited potent immunosuppressive effect, and it was possible to play important roles in colonization and causing systemic infection in the host, while it was not responsible for the extensive virulence variation between strains. This study provides novel information for understanding the pathogenesis of H. parasuis.更多还原