Isolation and Characterization of the 34 Kd Gene of Mycobacterium Leprae.
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Date
1993-09
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Addis Ababa University
Abstract
An M. Iprae::pHC79 cosmid genomic library was screened for the
identification, isolation and characterization of the 34 kd antigen gene of
Mycobacterium leprae using its homologue, the 34 kd antigen gene of
Mycobacterium tuberculosis, as a DNA probe. Qut of a total of 79 different
cosmids screened, covering about 88 % of the genome of M. leprae, one cosmid
designated as BL 1527 was found to harbor the gene of interest. Restriction enzyme
analysis of the cosmid BL 1527 yielded four DNA fragments - two containing the
complete gene and two containing part of the gene. The four DNA segments were
subcloned in plasmid vectors pUCBM21 and pBKS+ and characterized physically
and immunologically. Physical characterization of the gene by nucleotide
sequencing revealed that the 34 kd antigen gene has an open reading frame of 801
nucleotides encoding 266 amino acid residues bearing a molecular weight of28,887
daltons and an isoelectric point of 4.3. Homology comparison with the 34 kd
antigen gene from M.tuberculosis has revealed 80.6 % (633 nucleotides) identity
at the DNA level and 85 % (221 amino acids) identity at the protein level.
Immunological characterization by western blot analysis has shown that the gene
is expressed in E. coli from its own promotor signals and that the recombinant
protein is recognized by the monoclonal antibody F-126. 2 which is specific for the
mycobacterial 34 kd antigens. Crossed Immuno-electrophoresis analysis has
established that the recombinant 34 kd antigen corresponds to the non secreted
Antigen 84 of mycobacteria.
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Biology