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| Author(s): |
Andressa Caravelli
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2013-04-12 |
| Examining board members: |
Roxane Maria Fontes Piazza;
Marilis do Valle Marques;
Jorge Luiz Mello Sampaio
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| Advisor: | Roxane Maria Fontes Piazza |
| Abstract | |
Intimin is the main virulence factor involved in the attaching/effacing lesion of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), which are responsible for infantile diarrhea and hemorrhagic colitis/hemolytic uremic syndrome, respectively. In developing countries, their diagnosis is either time-consuming and expensive or is not done in the routine laboratory. Nevertheless, their detection is essential in defining the infection treatment. The aim of the study was to define the most suitable expression vector and evaluate the sensitivity and specificity of the scFv-intimin obtained. The scFv-intimin gene previously obtained was cloned into pAE and pSMT3 expression vectors and transformed into an E. coli BL21 (DE3) strain. Expression was induced with 1 mM IPTG. In both vectors, recombinant protein was expressed as inclusion bodies, so pAE was chosen as the expression vector for nickel affinity chromatography. Once purified, scFv-intimin was employed in an indirect immunofluorescence assay to define a rapid and reliable assay testing 199 E. coli strains and other Enterobacteriaceae isolates, of which 132 isolates were eae-positive and 67 isolates were eae-negative. The immunofluorescence assay showed the detection of 132 eae-positive isolates (100% sensitive) and no detection in the 67 eae-negative isolates (100% specific). Thus, immunofluorescence is an effective and rapid method and scFv-intimin an excellent tool for the immunodiagnosis of EPEC and EHEC. (AU) | |
| FAPESP's process: | 11/14361-0 - Recombinant antibodies (scFv-intimin) as a tool for early detection of diarrhea caused by enteropathogenic Escherichia coli (EPEC) Escherichia coli and Shiga toxin producing (STEC). |
| Grantee: | Andressa Caravelli |
| Support Opportunities: | Scholarships in Brazil - Master |