PURIFICATION OF A MYCOBACTERIAL ADHESIN FOR FIBRONECTIN
PURIFICATION OF A MYCOBACTERIAL ADHESIN FOR FIBRONECTIN
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DOI：
10.1128/iai.61.5.1889-1894.1993
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发表时间：
1993-05-01
影响因子：
3.1
通讯作者：
BROWN, EJ
中科院分区：
文献类型：
--
作者：
RATLIFF, TL;MCCARTHY, R;BROWN, EJ
先前的研究表明分支杆菌附着在纤维连接蛋白（FN）上。分枝杆菌附着于FN被认为在牛分枝杆菌BCG治疗浅表性膀胱癌、启动对分枝杆菌抗原的迟发性超敏反应以及上皮细胞吞噬分枝杆菌中具有生物学重要性。因此，我们纯化了FN的分枝杆菌受体。母牛分枝杆菌培养3周后的培养上清液中含有结合FN并抑制两种M. vaccae和BCG至FN用作受体来源。冻干M.在0.02M bis-Tris（pH6.0）中重构母牛上清，并依次上样到ACA 54凝胶过滤柱和DEAE-Sephacel阴离子交换柱上。获得了纯化的55 kDa的抑制蛋白（p55）。观察到纯化的p55蛋白以剂量依赖性方式与FN结合并抑制I-125-FN与活BCG的结合。产生针对该蛋白的多克隆和单克隆抗体。得到的多克隆抗血清在粗M.通过Western印迹（免疫印迹）与55-kDa BCG蛋白交叉反应，并识别与BCG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M免疫的小鼠中分离单克隆免疫球蛋白M（λ）。疫苗p55蛋白在Western印迹中没有功能，但抑制活BCG与FN的附着。这些研究表明，M（r）为55，000的蛋白质或抗原相关蛋白质作为至少两种不同分枝杆菌的FN受体起作用。
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 I-125-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.