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Page No. : 13-18

ABSTRACT

An attempt was made in the present study to create a dominant phenotype of protective antigen (PA), which can block the lethal activities of native anthrax toxin by a rapid, cheap and efficient method named PCR based site-directed mutagenesis. The mutation was carried out in two step PCR reaction at positions 1279-1280so that phenyl alanine at 427th position was changed to aspartic acid. For this, the first PCR was carried out with mutation primer pairs viz., PA-F & mut PA-R and mut PA-F & PA-R, which amplified the required large fragment and small fragment from pX-O1 plasmid of B. anthracis strain 34F2. Then a second PCR was run in which products of the first PCR were employed as template and PA-F & PA-R as primers, so that the full length of pag containing desired mutation was amplified. Subsequently, the mutated pag was cloned into pJET. The positive clones were confirmed by colony PCR, plasmid PCR and sequencing. Nucleotide sequencing of recombinant plasmid revealed that the nucleotides at 1279-1280th positions were mutated without any other alterations in nucleotide sequence of pag gene. Now, further studies are required to express pag and confirm the therapeutic potential of this mutated PA against anthrax.

Keywords: Anthrax, protective antigen, site directed mutagenesis, therapy, vaccine