Wiker H G, Lyashchenko K P, Aksoy A M, Lightbody K A, Pollock J M, Komissarenko S V, Bobrovnik S O, Kolesnikova I N, Mykhalsky L O, Gennaro M L, Harboe M
Institute of Immunology and Rheumatology, University of Oslo, Norway.
Infect Immun. 1998 Apr;66(4):1445-52. doi: 10.1128/IAI.66.4.1445-1452.1998.
MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.
MPB70和MPB80（MPB70/80）以及MPB83是密切相关的抗原，它们在牛分枝杆菌中高度表达。MPB70/80是可溶性分泌抗原，而MPB83是与细菌表面相关的输出脂蛋白。在本研究中，这些抗原在还原和非还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中具有不同的迁移率。这些差异可能是由于MPB70和MPB83都有两个内部半胱氨酸残基，它们会通过二硫键形成环状结构。我们使用针对牛纯化蛋白衍生物或完整牛分枝杆菌细胞产生的单克隆抗体（MAb）分析了MPB70/80和MPB83的结构。单克隆抗体1-5C与MPB70和MPB80特异性反应，单克隆抗体MBS43与MPB83特异性反应，而其他抗体，包括几种先前描述的单克隆抗体，与所有三种抗原结合。单克隆抗体和多克隆抗体与还原蛋白反应强烈，与非还原蛋白反应较弱，表明线性表位的参与。通过使用MPB70的合成肽对单克隆抗体Bov-1、2-6B、1-5C和1-1D的表位进行了定位。序列比较显示，具有1-5C反应性表位的肽在三个残基上与MPB83同源区域中的残基不同。在第112位将A替换为S或在第116位将Q替换为E消除了单克隆抗体1-5C的反应性。针对天然纯化MPB70的多克隆兔抗体与成熟MPB70 N端一半的肽6、7和8强烈反应。实验性感染牛分枝杆菌动物的牛血清识别更广泛的肽谱。这些发现表明这些蛋白质具有诊断潜力，并且抗体在阐明涉及表面结合和暴露的脂蛋白MPB83及其高度同源的可溶性分泌MPB70/80对应物的宿主-分枝杆菌关系中也可能发挥作用。
