RESUMO
The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.
Assuntos
Anticorpos Monoclonais/biossíntese , Regulação da Expressão Gênica , Vetores Genéticos , Fragmentos Fab das Imunoglobulinas/biossíntese , Anticorpos Monoclonais/genética , Resistência ao Cloranfenicol/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli , Vetores Genéticos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.
Assuntos
Anticorpos Anti-Hepatite C/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Vacinas Virais/imunologiaRESUMO
It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD phenotype and can help elucidate the role played by natural antibodies in defence against infectious agents.
Assuntos
Anticorpos Antivirais/genética , Antígenos Virais/imunologia , Clonagem Molecular , Genes de Imunoglobulinas , Imunoglobulina D/genética , Imunoglobulina M/genética , Anticorpos Monoclonais/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Bacteriófagos/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Antígenos HIV/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Imunidade Inata , Imunoglobulina D/sangue , Imunoglobulina D/imunologia , Fragmentos de Imunoglobulinas/genética , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Biblioteca de Peptídeos , Plasmídeos/genéticaRESUMO
The preparation of random combinatorial libraries exposed on the surface of phage provides a route for the selection of diverse high affinity human monoclonal antibodies. However, in particular settings, the isolation of genes coding for a rare antibody can be elusive because some epitopes are predominant and because, in the case of impure antigens, the protein or any compound of interest can be present in relatively minimal amount. In this paper, we describe the successful utilization of a new strategy of "preadsorption" panning that allowed us to clone a rare human monoclonal antibody fragment and to access a different antibody repertoire. The procedure is easy, fast, inexpensive, can be used together with other panning techniques and can be particularly useful in cloning antibodies against rare or unknown determinants.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Clonagem Molecular , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Anticorpos Monoclonais/genética , Antígenos Virais/imunologia , Bacteriófagos , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Herpes Simples/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.