Cloning, Sequencing, and Expression of FanC Antigen of Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli (ETEC) strains are the major cause of diarrhea in new born calves and certain species such as humans, pigs and sheep. ETEC has the ability to bind with entrocytes through K99 and F41, and to produce enterotoxins as a result. The purpose of this study was to produce the recombinant FanC protein of Enterotoxigenic Escherichia coli in prokaryotic system by using pET32a(+) expression vector. Therefore, using specific primers and polymerase chain reaction, FanC(K99) gene was lified from Enterotoxigenic Escherichia coli. After purification, the gene was cloned and subcloned into pTZ57R/T and pET32a(+) respectively. The recombinant vector was transferred into E.oli BL21CodonPlus (DE3) as expression host, and the produced protein was purified by NiNTA chromatography column. The results of protein expression showed that E.coli was able to express FanC protein appropriately. This gene was expressed by the induction of IPTG at the concentration of 1mM. This was confirmed by Nindash NTA column, dotblotting analysis and SDSPAGE electrophoresis. The concentration of the recombinant protein was calculated as 1.3 mg/ml. The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant FanC protein. This recombinant protein may be suitable to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies.