Cloning and expression of gene FanC-2NT encoding K99-2NT fimbrial antigen of enterotoxigenic Escherichia coli from diarrheic post-weaning piglets

Abstract

Background and Purpose: The K99 (F5) is one pilus adhesin that mediates the attachment of enterotoxigenic E. coli (ETEC) strains to small intestines to cause to diarrhea in piglets, lambs and newborn calves. In this work, we carried out cloning and expression of the mature peptide of FanC subunit, K99 fimbriae, one of the most common adhesive antigens in E. coli. Materials and Methods: E. coli 2NT strain was isolated from fecal samples of post-weaning piglets with diarrhea. The coding sequence of the mature peptide of K99-2NT subunit was isolated by PCR amplification and cloned into pGEM®-T Easy vector for sequencing using fluorescent dideoxy-terminator method. Expression of K99-2NT protein which was inserted into pET200/D-TOPO vector induced with IPTG. The PCR product and expression level of protein was examined by agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Results and Conclusions: We cloned and expressed successfully the mature peptide of K99 subunit with molecular weight of approximately 17.5 kDa from E. coli 2NT strain (named K99-2NT). Nucleotide sequence of the K99-2NT subunit coding region of fanC-2NT gene is 477 bp in length and is 99% similarity with that of fanC gene (accession no: M35282). Highest expression level occurred after 12 h of induction with 0.75 mM IPTG at 37oC. This subunit antigen will be tested for immune response of rat in the next time