Cloning and sequence analysis of Mycobacterium bovis Ag85A gene

The genomic DNA was extracted from Mycobacterium bovis strain Vallee111. The mature secreted protein Ag85A gene was amplified with a pair of specific primers using polymerase chain reaction (PCR). PCR product was an approximate 900 bp DNA segment. The clone vector pGEM-T-85A was constructed successfully by the PCR product that was cloned into pGEM-T vector using T-A clone technique and recombinant clone was identified by using α-complementation test, plasmid size test, restrictional (enzyme) assay, plasmid PCR identification and recombinant plasmid sequence analysis. These results could serve as a basis for further studying on the usefulness of Ag85A gene and immunogenicity characteristic of Ag85A gene expression product .