RESUMEN
A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.
Asunto(s)
Clonación Molecular/métodos , Fragmentos Fab de Inmunoglobulinas/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Aminoácidos , Secuencia de Bases , Preescolar , ADN , Escherichia coli , Biblioteca Genómica , Humanos , Datos de Secuencia MolecularRESUMEN
We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.