Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells.

Abstract

Background:Flagellin is the main structural protein of the flagella of many pathogens including Salmonella typhimurium. It is a potent trigger of innate immune responses that enhance adaptive immune responses to a variety of protein antigens. Flagellin has intrinsic adjuvant activity mediated through toll-like receptor (TLR) 5 and is an attractive candidate for highly effective vaccine adjuvant conferring enhanced antibody and cellular immune responses to proteins or peptides. In the present study, we cloned the fliC gene from S. enterica typhimurium in eukaryote vector pVAX1 and evaluated its expression in eukaryotic cells.Objectives:The main aim of the present study was to construct a DNA vaccine expressing fliC as an adjuvant.Materials and Methods:The fliC gene of S. typhimurium (ATCC 14028) was amplified by PCR with specific primers and cloned into the pPrime cloning vector and successfully subcloned into expression vector pVAX1. The recombinant plasmid pVAX-fliC was finally expressed in eukaryotic cells.Results:Cloning and subcloning of the fliC gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pPrime-fliC and pVAX-fliC. The expression of flagellin protein in eukaryotic cells was approved by immunofluorescence assay (IFA), western blotting analysis and the reverse transcriptase polymerase chain reaction (RT-PCR) method.Conclusions:The results of this study demonstrated that the fliC gene in recombinant plasmid pVAX-fliC was successfully expressed in eukaryotic cells and produced flagellin protein, which could be used as an effective adjuvant for DNA vaccine research.