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

Abstract

BackgroundIntimin 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).FindingsAnti-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.ConclusionThis 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.

Diarrheagenic Escherichia coli

“…These authors clearly demonstrated that polyclonal rabbit antisera is suitable for immunoblotting as a diagnostic tool, and showed that protein denaturation and linearization is a critical step for anti-intimin antibody accessibility. Indeed, even employing the recombinant antibody such as single chain fragment variable (scFv-intimin),147, 148 merely by immunofluorescence the scFv-intimin was able to detect tEPEC, aEPEC, and EHEC isolates, showing that intimin can be a target for EPEC and EHEC diagnosis after bacterial permeabilization 148 …”

Anticorpo recombinante anti-intimina: uma ferramenta para o diagnóstico rápido de Escherichia coli enteropatogênica e de Escherichia coli enterohemorrágica.

“…Diversas abordagens vem sendo utilizadas para a produção de moléculas de scFv, bem como a possibilidade de interação destas moléculas com outras proteínas para tratamento e diagnóstico de doenças como o câncer, podem eficientemente localizar tumores e diminuir massas tumorais por bloquear estes antígenos para qual sua especificidade foi desenhada (BATRA et al, 1992;KETTELEBOROUGH et al, 1994;LEONG;CHEN, 2008;LORIMER et al, 1996;MAHGOUB et al, 2012;WEISSER;HALL, 2009 Essa menor reatividade do anticorpo monoclonal levou esses autores a produzir um anticorpo recombinante anti-intimina obtidos a partir da clonagem e expressão do fragmento variável de cadeia única (scFv) que reconheceu tanto a intimina purificada, como a intimina expressa pelo isolado protótipo de EPEC típica (MENEZES et al, 2011). …”

“…A figura 12 apresenta esquematicamente as etapas de amplificação das cadeias leve e pesada do fragmento variável do anticorpo monoclonal anti-intimina para posterior clonagem no vetor de expressão (MENEZES et al, 2011).…”

“…Com as sequências confirmadas, partiu-se para a montagem do scFv-intimina utilizando a sequência da nova cadeia leve, unida por um ligante peptídico à cadeia pesada obtida por Menezes et al (2011), como mostrado no quadro 6.…”

Clonagem e expressão de um fragmento variável em cadeia única contra a toxina termo-lábil de Escherichia coli enterotoxigênica.

“…FONTE: OZAKI, 2011. BASKHARAN et al, 2005;CHUNG et al, 2008;GOLCHIN;AITKEN, 2008;KHÜNE et al, 2004;MA et al, 2008;MENEZES et al, 2011).…”