Single Point Mutation as a Molecular Tool for Preparation of Recombinant Vaccine Against Caseous Lymphadenitis (CLA) and Oedematous Skin Disease (OSD) in Buffaloes

Abstract

The gene encoding the phospholipase D exotoxin (PLD) of Corynebacterium pseudotuberculosis (C. pseudotuberculosis); the causative agent of Caseous Lymphadenitis (CLA) in sheep and Oedematous Skin Disease (OSD) in buffaloes was mutagenised using PCR-site directed mutagenesis. Mutagenesis was performed to substitute Histidine codon 20 (CAC) with Tyrosine codon 20 (TAT) within the enzymatic active site of the pld gene. The wild and mutated pld genes were expressed in E. coli purified from cell lysate using calmodulin affinity chromatography (stratagene). A comparative study about synergistic hemolytic activity, dermonecrosis; immunogenicity and antigencity of both recombinants PLD (rPLD) was performed. Results showed that mutated recombinant PLD (mrPLD) had lost the synergistic hemolytic activity (SH), but both had the same characteristic in relation to dermonecrosis, immunogenic and antigenic activities. The results suggest that rPLD can be recommended as highly specific antigens for early diagnosis of CLA in small ruminants in addition to form the basis for recombinant PLD vaccines.

Key words: Wild recombinant PLD, mutated recombinant PLD, dermonecrotic activity, Immunogencity of rPLD, Mutagenesis.