CHARACTERIZATION OF MYCOBACTERIUM INTRACELLULARE LAMBDA-GT11 RECOMBINANT CLONES

弥散型M. avium和M.细胞内（MAC）疾病是一种流行的 艾滋病患者的机会性感染 不幸的是， MAC感染的诊断和临床管理在很大程度上 不满意。 努力制备可用于临床的MAC抗原 可用作特异性血清诊断或皮肤试验试剂或作为保护剂 免疫原也不成功。 此前，我们报道了一个M。 允许鉴定的胞内lgt11表达文库 并在E.杆菌 我们还 分离出5种不同的细菌， 来源于22、40、43、54和85的重组分枝杆菌蛋白 kDa M.胞内是天然抗原。 我们现在已经证明，粗E。 含有这些重组蛋白的大肠杆菌裂解物刺激T细胞 体外增殖。 我们还克隆了编码4种 将这些抗原导入高效表达载体pGEM中，并纯化， 通过电洗脱和HPLC过量生产蛋白质。 皮肤试验测定， 用这些纯化的蛋白质致敏的豚鼠已经证明 这些重组分子中的每一个都引起了一个重要的交叉， DTH反应 近几个月来，我们从我们的 表达文库，表达源自20， 34和70 kDa M.胞内抗原。 的免疫反应性 目前正在对这些多肽进行评估。 43 kDa M. 细胞内衍生的重组蛋白，其用 MAC特异性单克隆抗体IES与M.鸟 感染的小鼠，而不是对照鼠血清。 因为 这种蛋白质的免疫反应性，我们正在完成的DNA序列， 编码这种抗原的基因 此外，通过基因缺失和 表位作图实验，我们已经确定了DNA序列编码 MAC特异性B细胞表位。 多肽的免疫学评价 从基因序列推断应该允许一个确切的定义， MAC特异性B细胞表位及潜在T细胞的鉴定 单特异性皮肤测试分子。

Disseminated M. avium and M. intracellulare (MAC) disease is a prevalent opportunistic infection in AIDS patients. Unfortunately, early diagnosis and clinical management of MAC infections have been largely unsatisfactory. Efforts to prepare MAC antigens which may be clinically useful as specific serodiagnostic or skin-test reagents or as protective immunogens have also been unsuccessful. Previously, we reported on a M. intracellulare lgt11 expression library which permits the identification and expression of mycobacterial antigens in E. coli. We have also isolated 5 different bacterial phages which express genes for recombinant mycobacterial proteins derived from 22, 40, 43, 54 and 85 kDa M. intracellulare native antigens. We now have shown that crude E. coli lysates containing these recombinant proteins stimulate T cell proliferation in vitro. We have also cloned the genes encoding 4 of these antigens into the high expression vector, pGEM, and purified the overproduct proteins by electroelution and HPLC. Skin test assays in sensitized guinea pigs with these purified proteins have demonstrated that each of these recombinant molecules evokes a significant cross- reactive DTH reaction. In recent months, we have identified from our expression library, phages that express fusion proteins derived from 20, 34, and 70 kDa M. intracellulare antigens. The immunoreactivity of these polypeptides is currently being evaluated. The 43 kDa M. intracellulare-derived recombinant protein which was isolated with the MAC specific monoclonal antibody, IES, reacts with sera from M. avium infected mice and not with control murine sera. Because of the immunoreactivity of this protein, we are completing the DNA sequence of the gene encoding this antigen. Furthermore, by gene deletion and epitope mapping experiments we have determined the DNA sequence encoding the MAC-specific B cell epitope. An immunologic evaluation of peptides inferred from the gene sequence should permit an exact definition of the MAC specific B cell epitope and the identification of potential T-cell monospecific skin test molecules.