Resumo

Introduction: ETEC pathogenic strains produce heat-labile toxin (LT) and/or heat-stable toxin (ST) that differ in their structure and function while both are used as markers for detection of infections. The advances on antibody biotechnology provide alternatives to obtain low cost antibodies with desirable affinities and specificities by manipulating immunoglobulin domains, which consists in cloning immunoglobulin’s heavy and light variable domains (HV and LV) as a single-chain fusion interspaced by a flexible linker, therefore allowing the correct interaction between the domains and preserving the antigen-binding site. Objectives: Construction of a scFv upon hybridoma cells that produce an anti-LT monoclonal antibody following its bacterial production. Methods: After RNA extraction from hybridoma cells and reverse transcription, coding regions of heavy and light chain variable domains were PCR-amplified and fused to a linker giving rise to the scFv-LT coding region. The DNA construct was cloned into p-GEM-T Easy vector that was used as template for sequencing. A synthetic gene was generated based on this sequence and subcloned into pET28a vector. The new recombinant plasmid was used to transform competent E. coli BL21(DE3) cells. Transformed cells were cultured until reach 0,6 OD_{600 nm}. After induction by IPTG, the cells were harvested and disrupted by pressure. The inclusion bodies were isolated, solubilized with 8M urea buffer and refolded by dilution. The refolded proteins were submitted to metal affinity chromatography using Ni-NTA resin and step-wise elution. Fractions were analyzed by SDS-PAGE.  The recognition of LT by scFv-LT was tested by ELISA. Results/Discussion: The amplification of VH, VL and scFv-LT showed fragments containing 325 bp, 340 bp and 723 bp, respectively. The target protein was identified as a major band with apparent molecular weight 30 kDa, with no biochemical activity. The design of synthetic gene was necessary to optimize the expression of recombinant protein in bacteria. The expression of the transcript presented expected size, although with no biological activity. The lack of activity could be explained by uncorrected protein fold. Other refolding method or expression strains should be used to obtain a molecule with biological activity. Once obtained this molecule could be used as a tool for ETEC detection.