RESUMO
Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Geminiviridae/genética , Região Variável de Imunoglobulina/metabolismo , Vírus do Mosaico/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Clonagem Molecular , Drosophila/genética , Drosophila/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Geminiviridae/imunologia , Humanos , Immunoblotting , Região Variável de Imunoglobulina/genética , Vírus do Mosaico/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage. Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes. The binding specificities were confirmed by Western blots and ELISA. The scFv preparations reacted with B. napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli. Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule. The scFv were used to detect ENR directly in extracts of B. napus seeds.
Assuntos
Brassica/enzimologia , Fragmentos de Imunoglobulinas/imunologia , Oxirredutases/análise , Oxirredutases/imunologia , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Bacteriófagos/genética , Western Blotting , Clonagem Molecular , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Graxo Sintases/análise , Ácido Graxo Sintases/imunologia , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sementes/enzimologia , Alinhamento de SequênciaRESUMO
A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.
Assuntos
Fosfatase Alcalina/imunologia , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fragmentos de Imunoglobulinas/imunologia , Luteovirus/isolamento & purificação , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Anticorpos Antivirais/genética , Antígenos Virais/análise , DNA Viral/análise , Humanos , Fragmentos de Imunoglobulinas/genética , Luteovirus/imunologia , Dados de Sequência Molecular , Extratos Vegetais , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Solanum tuberosum/virologiaRESUMO
Coat protein gene sequences of eight isolates of potato mop-top virus from the Peruvian Andes and of three isolates from Scotland were compared. Despite wide geographical separation, there was little sequence variation among all isolates.
Assuntos
Capsídeo/genética , Sequência Conservada , Vírus de Plantas/genética , Vírus de RNA/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Dados de Sequência Molecular , Vírus de Plantas/isolamento & purificação , Homologia de Sequência de Aminoácidos , Solanum tuberosum/virologiaRESUMO
Antibody fragments (scFv) that bind specifically to particles of cucumber mosaic cucumovirus (CMV) were obtained from a library which encodes a diverse array of synthetic antibody fragments, each displayed on the surface of filamentous bacteriophage. After four rounds of selection and enrichment, several clones were obtained which produced scFv that bound specifically to purified particles of CMV in ELISA. BstNI digestion of phagemid DNA resulted in the same restriction pattern for all clones. The nucleotide sequences of three of the clones showed that they belonged to the human VH1 family and that they had a complementarity determining region loop of 7 amino acids. Phage-displayed antibodies and soluble scFv secreted by these clones reacted with particles of CMV in sap from infected plants in ELISA. In immunoblotting tests, soluble scFv preparations reacted with SDS-denatured coat protein extracted from purified preparations of CMV isolates belonging to either subgroup I or II and also with protein extracted by SDS treatment of seeds harvested from naturally infected lupin plants. The results demonstrate the feasibility, and potential applicability, of recombinant antibody methods in plant pathology.
