Cloning of Mycoplasma agalactiae P40 gene in prokaryotic system

The P40 gene encodes adhesion protein that is expected to be involved in adhesion of M. agalactiae to eukaryotic cell and promote host colonization. Lipoprotein P40 is an immunodominant antigen and a vaccine candid. The target of this study is recombinant subunit vaccine, so P40 gene was cloned in prokaryotic system. In this study, the P40 gene of three vaccinal isolated M. agalactiae strains in Iran was amplified and sequenced. After changes,the selected sequence of P40 gene was synthesized and cloned in cloning plasmid, then transformed into Escherichia coli by heat shock protocol. After amplification, P40 gene was digested by two enzymes. Purified gene was cloned in expression plasmid for transforming into expression bacterial cell. The results showed that 920 bp fragment of P40 genes from three vaccinal strains was amplified, so three strains have P40 gene. Complete sequence of this gene was 109 bp, was extracted from cloning plasmid and cloned in pET22b expression plasmid including T7 promoter and terminator. Finally, recombinant plasmid was transformed into bacterial cell. P40 gene cloning was first step in recombinant subunit vaccine production.