Background: High efficacy of subunit vaccine requires immunostimulant as an adjuvant component. Flagellin protein (FliC) in bacteria is considered as a potential candidate. However, the high antigenic property of FliC has made it difficult to apply for a variety of vaccine development. In this present study, we assessed the immunostimulant of FliCΔ220-320-a less immunogeneicity variant of FliC. In which, 100 amino acids were deleted from the hypervariable domain of FliC in fusion with GFP (plays as a model antigen). Methods: To generate this contract, we isolated FliC gene from Salmonella enterica Enteritidis (S. enteritidis) serving for cloning and expressing of FliC-delta220-320 (FliC∆220-320-gfp) protein in E. coli. The gfp gene was cloned into pET28a-FliCΔ220-320 resulting in a recombinant vector pET28a-FliCΔ220-320-gfp. The expression of FliC∆220-320-gfp protein was induced by 0.1 mM IPTG and confirmed by the SDS-PAGE analysis and Western blot probed with anti-6xHis antibody. This recombinant protein was then validated for immunostimulatory via subcutaneous immunization in mice. Results: FliCΔ220-320-gfp protein was expressed in soluble form and was purified by using the immobilized-metal affinity chromatography with a purity of 56%. The present results showed that the FliCΔ220-320 variant in fusing with GFP reduced its antigenic stimulation to four times than that of FliC in the FliC-gfp and retained its ability to stimulate a humoral immune response against fused GFP. Conclusion:This result suggested that it was possible to apply the FliCΔ220-320 variant to subunit vaccines in a form of fusion with protein antigen(s) to increase their efficacy.
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