Romain F, Laqueyrerie A, Militzer P, Pescher P, Chavarot P, Lagranderie M, Auregan G, Gheorghiu M, Marchal G
Unité de Physiopathologie de l'Infection, Institut Pasteur, Paris, France.
Infect Immun. 1993 Feb;61(2):742-50. doi: 10.1128/iai.61.2.742-750.1993.
Increased protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by a previous immunization with living avirulent mycobacteria, usually Mycobacterium bovis BCG. Only a transient and marginal protection is obtained after immunization with bacterial extracts or dead bacteria. Both living and heat-killed bacteria share a number of common antigens. In order to identify mycobacterial molecules which are dominant antigens during immunization with living bacteria, a two-step selection method was used. Two groups of guinea pigs were immunized either with living or with heat-killed BCG. Sera were then collected and used to select and counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride sheets. The molecules present on twin immunoblots were stained with antibodies raised in guinea pigs immunized either with living or with heat-killed BCG. Cross-reactive antigens stained in twin immunoblots were eliminated. Major antigens interacting with antibodies raised after immunization only with living bacteria were further purified. A complex of 45- and 47-kDa major molecules (45/47-kDa complex) was thus identified and further purified. The complex was found to interact only with antibodies present in sera of guinea pigs immunized with living bacteria and not at all with antibodies raised after immunization with dead bacteria. The 45/47-kDa antigen complex molecules were resolved on two-dimensional electrophoresis in three major and seven minor proteins detected with silver staining. All the molecules interacted with the antibodies present in sera of guinea pigs immunized with living BCG. The three major proteins (two at 47 kDa and one at 45 kDa) were amino-terminal sequenced. The sequence A-P-E-P-A-P-P-V-P-P-A-A-A-A-P-P-A, which was not previously reported, was the same for these three molecules. By using a competitive enzyme-linked immunosorbent assay, the concentrations of the 45/47-kDa antigen complex were measured in BCG culture filtrates, freeze-dried BCG, and dried heat-killed BCG; they were, respectively, 2, 0.01, and 0.001% of the total mass. The low or very low values compared with the high antibody concentration emphasized the ability of the 45/47-kDa complex delivered through live BCG to trigger an antibody response.
针对结核分枝杆菌强毒株攻击的增强保护作用主要由先前用减毒活分枝杆菌免疫诱导产生，通常是牛分枝杆菌卡介苗（Mycobacterium bovis BCG）。用细菌提取物或死菌免疫后仅能获得短暂且微弱的保护。活细菌和热灭活细菌都有许多共同抗原。为了鉴定在用活细菌免疫期间起主要抗原作用的分枝杆菌分子，采用了两步选择法。两组豚鼠分别用活卡介苗或热灭活卡介苗免疫。然后收集血清，用于选择和反选卡介苗培养滤液中存在的抗原。从一系列高压液相色谱柱（凝胶过滤、二乙氨基乙基纤维素柱和反相色谱柱）洗脱的每个主要组分在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上进行电泳，并转移到聚偏二氟乙烯膜上。用用活卡介苗或热灭活卡介苗免疫的豚鼠产生的抗体对双免疫印迹上存在的分子进行染色。消除在双免疫印迹中染色的交叉反应性抗原。进一步纯化仅与用活细菌免疫后产生的抗体相互作用的主要抗原。由此鉴定并进一步纯化了一种由45 kDa和47 kDa主要分子组成的复合物（45/47 kDa复合物）。发现该复合物仅与用活细菌免疫的豚鼠血清中存在的抗体相互作用，而与用死细菌免疫后产生的抗体完全不相互作用。45/47 kDa抗原复合物分子在二维电泳上分离，银染检测到三种主要蛋白和七种次要蛋白。所有这些分子都与用活卡介苗免疫的豚鼠血清中存在的抗体相互作用。对三种主要蛋白（两种47 kDa和一种45 kDa）进行了氨基末端测序。这三种分子的序列A - P - E - P - A - P - P - V - P - P - A - A - A - A - P - P - A是相同的，此前未见报道。通过竞争性酶联免疫吸附测定法，测定了卡介苗培养滤液、冻干卡介苗和干热灭活卡介苗中45/47 kDa抗原复合物的浓度；它们分别占总质量的2%、0.01%和0.001%。与高抗体浓度相比，这些低或极低的值强调了通过活卡介苗递送的45/47 kDa复合物引发抗体反应的能力。
