RESUMO
Purpose: P. aeruginosa is the epitome of an opportunistic pathogen of humans. It causes a variety of serious illness and represents a major risk among patients with cystic fibrosis, hospital-acquired infections, especially in burn, and otherwise immunocompromised patients, including transplantation and cancer patients. This microbe has a high level of innate antimicrobial resistance and a large armamentarium of virulence factors that have been the subjects of vaccine development. Flagellum is one Of these virulence factors that play a major role in the recognition of bacteria by the host and in the induction of immune responses by Toll-like receptor 5[TLR5]. These data support Flagellin as an antigen candidate to design an anti -Flagellin vaccine. Isolation, cloning, expression and purification of Flagellin were aimed at in this study
Methods: flagellin gene sub type b fliC [B] was amplified by PCR from genomic DNA isolated from P. aeruginosa PA01 and then cloned into the cloning vector pCEMT Easy. Sub cloning of the amplified gene into expression vector pRSET-B. The recombinant Flagellin was expressed after transformation into E. coli BL-21 [DE3] pLysS and finally purified using Ni+2 affinity chromatography
Results: a 1460 bp fragment of the fliC [B] gene from P. aeruginosa strain PAO1 was amplified from genomic DNA by PCR. This fragment was cloned into the TA cloning vector pGEMT-Easy and after transformation into E. coli DH5 alpha, selection of the clones was carried by blue-white screening. Confirmed positive plasmid extracted from some white colonies was digested, gel purified and ligated to pRSET- B expression vector that has also been digested and gel purified. This recombinant vector was transformed into E. coli DH5 alpha and some white colonies were picked and miniprepped to screen for the presence of the insert by restriction digests. Plasmids with the fliC [B] insert were used for expression of fliC [B] by transformation into E. coli BL-21 [DE3] pLysS. SDS-PAGE electrophoresis confirmed the expression of target protein having molecular weight of 53 KDa
Conclusion: our findings confirmed that a prokaryotic expression system of recombinant fliC [B] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pRSET-B /fliC [B] / BL-21 [DE3] efficiently produced target recombinant protein in the form of soluble protein. Therefore we can suggest that this purified protein can effectively be a vaccine candidate