title: Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv).
abstract: BACKGROUND
  Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).
  
  
  RESULTS
  Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.
  
  
  CONCLUSIONS
  This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.
authors.affiliation: Laboratório de Bacteriologia, Instituto Butantan, Avenida Vital Brazil, 1500, São Paulo, SP, 05503-900, Brazil. roxane@butantan.gov.br.
authors.forename: Márcio A
authors.initials: MA
authors.lastname: Menezes
authors.forename: Karina A
authors.initials: KA
authors.lastname: Aires
authors.forename: Christiane Y
authors.initials: CY
authors.lastname: Ozaki
authors.forename: Renato M
authors.initials: RM
authors.lastname: Ruiz
authors.forename: Milton Ca
authors.initials: MC
authors.lastname: Pereira
authors.forename: Patrícia Ae
authors.initials: PA
authors.lastname: Abreu
authors.forename: Waldir P
authors.initials: WP
authors.lastname: Elias
authors.forename: Oscar Hp
authors.initials: OH
authors.lastname: Ramos
authors.forename: Roxane Mf
authors.initials: RM
authors.lastname: Piazza