The broad, long-term objectives of the proposed research are to identify new Mycobacterium leprae (M. leprae) proteins in an effort to expand our knowledge about the growth, regulation, and antigenicity of this pathogenic organism. Awareness of the disease leprosy dates to biblical times yet the causative agent, M. leprae, is one of the last remaining infectious disease pathogens to be fully characterized antigenically. The evasiveness of M. leprae results from both our inability to culture the mycobacteria for prolonged periods of time and its limited host range. When organisms are recovered from infected tissues, sonicated, and subjected to polyacrylamide gel electrophoresis there is an abundant number of proteins demonstrated, yet relatively few of these 30 plus proteins have been identified and characterized. More information about M. leprae proteins is important for vaccine development, studies of the pathogenesis of the organism, and diagnostic assays. This project focuses on bacteriophage clones that we have isolated from a lambdagtll expression library of M. leprae that were reactive by screening with hyperimmune rabbit antisera.
The specific aims are to: 1) Probe a cosmid library of M. leprae DNA with a gene fragment corresponding to the carboxyl terminus of a 64kDa protein of M. leprae that we have recently identified so that we can obtain the complete DNA sequence of this gene. 2) Sequence the entire M. leprae DNA insert that contains the 3' end of the gene for the 64kDa protein in order to determine if additional open reading frames or repetitive elements are present in the large DNA fragment. 3) Run computer analyses on the nucleotide sequences generated to provide information on possible biochemical function(s). 4) Determine if the 64kDa protein is important in the humoral immune response of humans by Western blotting and indirect enzyme-linked immunosorbent assays. 5) Screen the additional clones that we have isolated from the lambdagtll library with newly described monoclonal antibodies and determine the size of the M. leprae proteins that are represented by these clones. 6) Subclone and sequence the M. leprae DNA from these reactive clones if the antigenic determinants they express are non-reactive with monoclonals. 7) Train undergraduate students in modern molecular biology techniques used in studying and isolating DNA and proteins.
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