Construction of S1 pertussis toxin mutant gene for induction of homologous recombination in Bordetella pertussis

Despite high vaccination coverage, Pertussis has re-emerged in several countries and also in Iran. Antigenic divergence due to virulence genes polymorphism between circulating and vaccine strains was proposed as the main contributory factor. Pertussis toxin (PTX) is an important virulence factor produced by Bordetella pertussis. The toxic activity of PTX relies onan enzymatic subunit S1 (ptx-S1) having ADP-ribosylating activity. Herein we describe the construction of an inactivemutant ptx-S1 cassette for induction of hemolegous recombination in regional B.pertussis strains.Based on ptx heterogeneity, the dominant variant among Iranian B.pertussis strains has been selected for gene isolation andmutation. PTX-S1, toxin enzymatic domain, was amplified using specific primers and cloned into KpnI/BamHI site ofpUC18. The mutant cassette, pUCptx-S1M, contains R9K and E129G substitutions in enzyme active site was constructed using mutant primers and confirmed via sequencing.The upstream and downstream regions of ptx-s1, respectively 1287bp by ScaI/KpnI and 1531bp by BamHΙ/Hindшrestriction sites were amplified and cloned into pUC18 resulted pUC53.The S1M segment was extract from pUCptx-S1M by digestion and cloned into pUC53 between KpnI/BamHI sites. Eventually the mutant induction construct was cloned into B.pertussis specific shuttle vector pSS1129 and confirmed usingrestriction enzyme analysis and sequencing. The prepared construct will be applied for induction of homologous recombination in selective strain. Site direct mutagenesis is an effective strategy for residue replacement. Inactivation of PTX not only attenuates the toxin activity but also maintains the putative immunogenic factor.

کلیدواژه ها:

B.pertussis ، pertussis toxin ، Site direct mutagenesis