Olsen I, Reitan L J, Holstad G, Wiker H G
National Veterinary Institute, The National Hospital, Oslo, Norway.
Infect Immun. 2000 Feb;68(2):801-8. doi: 10.1128/IAI.68.2.801-808.2000.
Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.
通过交叉免疫电泳（CIE）以及使用鸟分枝杆菌鸟亚种多克隆多价抗血清吸收交叉反应性抗原，鉴定出了副结核分枝杆菌亚种的特征性抗原。两种抗原存在于副结核分枝杆菌亚种中，而在鸟分枝杆菌亚种中未检测到。在副结核分枝杆菌亚种抗原的CIE参考系统中，它们被鉴定为抗原17和20。纯化的抗原20被鉴定为烷基过氧化氢还原酶C（AhpC），而纯化抗原17的N端部分与结核分枝杆菌的烷基过氧化氢还原酶D（AhpD）具有80%的同源性。AhpC在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中具有非还原迁移率，对应分子量为45 kDa，其天然形式可能是通过二硫键连接的同型二聚体。AhpD的迁移率对应19 kDa。抗AhpC和AhpD的单特异性兔抗血清与9株副结核分枝杆菌亚种反应，但与其他20种分枝杆菌菌株无反应，除了一株戈登分枝杆菌菌株，该菌株产生了一条弱交叉反应带。实验感染副结核分枝杆菌亚种的山羊对AhpC和AhpD均有强烈的γ干扰素（IFN-γ）反应，并且它们也有抗AhpC的抗体。AhpC和AhpD诱导IFN-γ产生的能力表明，这些蛋白质未来有可能用于疫苗或诊断检测。这些结果进一步表明，AhpC和AhpD是免疫重要蛋白，在副结核分枝杆菌亚种中组成性且高表达，而细菌未受到氧化应激，并且抗原的特异性可能是不同物种中蛋白质表达水平不同以及结构差异明显的问题。
