The polymerase chain reaction (PCR) based on the selective amplification of a 530-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied on sections of fixed or frozen biopsy samples from leprosy patients. A simple procedure for the extraction of DNA from M. leprae in clinical specimens that provided suitable template DNA for amplification was developed. When PCR was applied on frozen sections, positive amplification in samples from all untreated acid-fast bacillus (AFB)-positive patients and in samples from 56% of the untreated AFB-negative patients could be detected, while biopsy samples from patients with skin diseases other than leprosy were all PCR negative. With neutral Formalin-fixed biopsy samples, positive amplification in 92% of the samples from untreated AFB-positive patients and in 61% of the samples from untreated AFB-negative patients could be detected by PCR. Biopsy samples exposed to mercuric chloride or nonbuffered formaldehyde containing fixatives were not suitable for application of PCR. This PCR holds promise as a tool for studies on M. leprae infection.
The 36,000-molecular-weight antigen (36K antigen) of Mycobacterium leprae is a major immunogenic protein carrying common and specific antigenic determinants recognized by antibodies and T cells in leprosy patients. Recombinant DNA clones containing the complete gene coding for the 36 K antigen, designated in this paper as PRA, were isolated from both lambda gtll and cosmid libraries of the M. kprae genome. The DNA sequence of the pra gene coded for a polypeptide of 249 amino acids with a predicted molecular mass of 26,299 daltons. The deduced amino acid sequence revealed a proline-rich (42%) amino-terminal region containing a number of repeated sequences similar or identical to the sequence PGGSYPPPPP. The reactivity of four monoclonal antibodies (F47-9, F67-1, F67-5, and F126-5) was directed to this proline-rich region of the PRA protein. DNA sequence and immunological data indicated that the lambda gtll recombinant Y3180, which was previously isolated by using antibody F47-9 (RNature (London) 316: [450][451][452] 1985), specffies a fusion protein unrelated to PRA but containing a similar epitope recognized by F47-9. Although Mycobacterium leprae was one of the first human pathogens to be described, relatively little is known about the components that play a role in the immunopathology of leprosy. The availability of purified and well-characterized antigens is a prerequisite for the determination of the role of individual molecules in the pathogenesis of leprosy and in the humoral and cellular immune responses to M.
Using synthetic peptides representing overlapping sequences of the 100-amino-acid-long N-terminal region of the proline-rich antigen of Mycobacterium leprae (PRA), we have mapped the epitopes in the primary structure of PRA recognized by four monoclonal antibodies. The M. Ieprae-specific monoclonal antibody F47-9 recognized the amino acid sequence LGSAYP (residues 34 to 39). Both monoclonal antibodies F67-1 and F67-5 recognized the sequence YPPP within the repeated sequence of PRA at four sites (residues 38 to 41, 50 to 53, 60 to 63, and 70 to 73). Monoclonal antibody F126-5 recognized the sequence SYPPP, also within the repeat, at three sites (residues 49 to 53, 59 to 63, and 69 to 73). All three epitopes appeared to be linear as far as can be determined by this approach.
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