Cloning and expression of immunogenic Clostridium botulinum C2I mutant proteins designed from their evolutionary imprints

Publication date: Available online 19 January 2019Source: Comparative Immunology, Microbiology and Infectious DiseasesAuthor(s): A. Prisilla, P. ChellapandiAbstractC2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2 T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298 F, S347 A and S350 A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298 F, S347 A and S350 A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced AD...
Source: Comparative Immunology, Microbiology and Infectious Diseases - Category: Infectious Diseases Source Type: research