CHARACTERIZATION OF MYCOBACTERIUM INTRACELLULARE LAMBDA-GT11 RECOMBINANT CLONES

点击翻译按钮获取中文摘要

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.