Ratliff T L, McCarthy R, Telle W B, Brown E J
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri.
Infect Immun. 1993 May;61(5):1889-94. doi: 10.1128/iai.61.5.1889-1894.1993.
Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria.
先前的研究表明，分枝杆菌可附着于纤连蛋白（FN）。在卡介苗治疗浅表性膀胱癌、引发对分枝杆菌抗原的迟发型超敏反应以及上皮细胞对分枝杆菌的吞噬作用中，分枝杆菌与FN的附着被认为具有重要生物学意义。因此，我们纯化了FN的分枝杆菌受体。将含有能结合FN并抑制母牛分枝杆菌和卡介苗与FN附着的蛋白质的母牛分枝杆菌3周培养物的培养上清液用作受体来源。冻干的母牛分枝杆菌上清液用0.02 M双三羟甲基氨基甲烷（pH 6.0）复溶，然后依次应用于ACA 54凝胶过滤柱和DEAE - 琼脂糖离子交换柱。获得了一种55 kDa的纯化抑制蛋白（p55）。观察到纯化的p55蛋白可与FN结合，并以剂量依赖的方式抑制125I - FN与活卡介苗的结合。制备了针对该蛋白的多克隆抗体和单克隆抗体。所得多克隆抗血清在母牛分枝杆菌粗提上清液中可印迹出一条55 kDa的单一蛋白带，通过蛋白质印迹法（免疫印迹）与一种55 kDa的卡介苗蛋白发生交叉反应，并识别出一条与卡介苗细胞壁相关的55 kDa条带，这与其作为FN受体的功能一致。从用纯化的母牛分枝杆菌p55蛋白免疫的小鼠中分离出一种单克隆免疫球蛋白M（λ），该蛋白在蛋白质印迹中无功能，但可抑制活卡介苗与FN的附着。这些研究表明，一种分子量为55,000的蛋白质或抗原相关蛋白可作为至少两种不同分枝杆菌的FN受体。
