Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Bābul (Feb 2017)
Cloning and Expression of Mutant and Inactive Form of Bordetella Pertussis Toxin from Iranian Native Strain
Abstract
BACKGROUND AND OBJECTIVE: Whooping cough is an acute contagious human respiratory disease especially in infants and children and is one of the ten major causes of death from infectious diseases. Despite reducing the risk due to vaccination with killed bacteria pathogen (Bordetella pertussis), increased disease emerged in recent decades indicates re-emerging due caused by genetic diversity of bacteria and the insufficiency of current vaccines. Therefore, the design of new and effective vaccines based on common strains of the population is essential. In this study the prevalent strain Iranian B.pertussis strains has been selected for gene amplification of pertussis toxin (PTX), the most important antigen of bacteria. The toxicity of the PTX related to its enzymatic activity therefore by site directed mutagenesis, the effective residues in the active site of the enzyme has been changed and the inactive and safe recombinant was produced. METHODS: Bacterial strain used in this study to determine the genome and gene amplification was selected from pathogenic strains isolated from Iranian patients from the pertussis reference laboratory of Microbiology of the Pasteur Institute of Iran. Genomic DNA was extracted by kit, ptx- s1(the enzymatic fragment of PTX) fragments amplified and inserted in pUC18. Site directed mutagenic primers were used for substitution of Arg 9 by Lys and Glu129 by Gly. The confirmed mutant ptx- s1 (mptx-s1) was sub-cloned into expression vector pET15b. The expression of recombinant protein in E.coli BL21 was induced by 0.5mM IPTG. The expression was analyzed by SDS-PAGE and immuno-blotting. The protein was puifird by Ni-NTA affinity chromatography under denature condition. FINDINGS: Sequencing data and restriction enzyme analysis confirmed the gene constructing and mutations. The presence of a 28 kDa band in SDS-PAGE and immunoblot showed the expression of mutant PTX. The concentration of recombinant protein was 0.36 mg/ml. CONCLUSION: The Recombinant protein which was obtained in this study provided the possibility of designing acellular vaccines on the basis of important antigens of prevalent strain of B.pertussis in Iran.
