Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

<p>Abstract</p> <p>Background</p> <p>Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic <it>Escherichia coli </it>(EPEC) and enterohemorrhagic <it>Escherichia coli </it>(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 (int_388-667). 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).</p> <p>Findings</p> <p>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 <it>pGEM-T Easy </it>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. <it>E. coli </it>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.</p> <p>Conclusion</p> <p>This study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int_388-667) in purified form and the EPEC isolate.</p

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Determination of tangential and normal components of oral forces

Oral forces applied to human teeth during biting and mastication are normally described in the literature only in terms of their axial components. The purpose of this study was to fully determine the spatial characteristics of the oral resultant force - its normal and tangential components - for a given individual. A load cell was especially manufactured to measure oral force and was temporarily implanted as a prosthetic device in the dental arch of a volunteer, replacing his missing upper first molar. The mastication and occlusion tests were carried out in such a way the cell should withstand the loads applied to the molar, and its state of strain was recorded by strain gauges attached to it. Based on the results of these tests and using balance equations, normal and tangential components of the resultant oral force were determined. For direct occlusion, without interposition any obstacle between cusps, a peak normal force of 135 N was recorded simultaneously to a tangential force of 44 N. For mastication of biscuits, a peak normal force of 133 N and a tangential force of 39 N were obtained