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Doctoral Thesis
DOI
Document
Author
Full name
Márcio Anunciação Menezes
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Piazza, Roxane Maria Fontes (President)
Fernandes, Irene
Maranhão, Andrea Queiroz
Martinez, Marina Baquerizo
Soares, Irene da Silva
Title in Portuguese
Anticorpos anti-intimina: análise da reatividade dos anticorpos policlonal e monoclonal, clonagem e expressão do fragmento variável de cadeia simples (scFv) do anticorpo monoclonal
Keywords in Portuguese
Anticorpo monoclonal
Anticorpos monoclonais (Avaliação
Bactérias patogênicas
Diarreia
EHEC
EPEC
Escherichia coli
Especificação)
Intimina
scFv
Abstract in Portuguese
Intimina é o principal fator de virulência envolvido na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecção de EHEC e EPEC típica ou atípica é de fundamental importância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos países desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentaram 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 µg desse anticorpo reconheceu 0,6 µg de intimina purificada com uma constante de dissociação de 1.3 x 10-8 M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isso, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplificadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressão pAE. Linhagem de E. coli BL21(DE3)pLys foi transformada com o plasmídeo pAE-scFv antiintimina e submetida à indução protéica. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteína por 100 mL de cultivo bacteriano. Para testar a funcionalidade do scFv, foram realizados ensaios de ELISA de captura e imunofluorescência. Os resultados mostraram que 275 ng de scFv reagiram com 2 µg de intimina purificada a uma absorbância de aproximadamente 0,75 e por imunofluorescência mostrou uma forte reatividade ao isolado de EPEC típica E2348/69. Este estudo demonstrou que o anticorpo recombinante anti-intimina obtido foi capaz de reconhecer a região conservada de intimina (Int388-667) na forma purificada e a intimina α no isolado de EPEC típica, e se mostrou mais eficiente que o anticorpo monoclonal nativo.
Title in English
Anti-intimin antibodies: polyclonal and monoclonal reactivity analyzes, cloning and expression of single chain fragment variable (scFv) of monoclonal antibodies
Keywords in English
Diarrhea
EHEC
EPEC
Escherichia coli
Intimin
Monoclonal antibody
scFv
Abstract in English
Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections caused by E. coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificity of polyclonal and monoclonal antibodies against intimin in the detection of EPEC and EHEC by immunoblotting. All employed antibodies showed 100% specificity and the sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, respectively. This anti-intimin monoclonal was characterized as IgG2b and 1 mg recognized 0.6 µg of purified intimin with a dissociation constant of 1.3 x 10-8 M. The less extent reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFv). Thus, the anti-intimin hybridoma mRNA was extracted, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in a strategy to amplify and link the chains, obtaining the scFv, which was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction of protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed. The results showed that 275 ng of scFv reacted with 2 µg of purified intimin resulting in an absorbance of 0.75. By immunofluorescence it was observed a strong reactivity to the typical EPEC isolate E2348/69. This study demonstrated that the recombinant anti-intimin antibody obtained was able to recognize the conserved region of intimin (Int388-667) in its purified form and α intimin in a typical EPEC isolate, and was more efficient than the native monoclonal antibody.
 
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Publishing Date
2011-06-21
 
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