RESUMEN
We have combined the high cloning efficiency of the lambda bacteriophage vectors with the surface expression screening method for the display of combinatorial antibody fragment (Fab) libraries on the surface of filamentous phage particles. The utility of the herein described ImmunoZAP 13 system for the isolation of Fabs that specifically bind antigen is demonstrated using two phagemid display libraries prepared from a previously characterized human combinatorial library. The percentage of clones that specifically bind antigen is maintained throughout the process of subcloning the LC and VH genes into ImmunoZAP 13, in vivo mass excision to convert the lambda library to a phagemid library, and preparation of phagemid particles displaying Fabs. Specific phagemid were isolated from libraries containing 0.6% and 0.03% tetanus toxoid (TT)-binding clones after two and three rounds of biopanning, respectively. Relative binding curves determined on a small sample of isolated clones indicate that several unique immunoglobulin Fab fragments have been isolated.
Asunto(s)
Bacteriófago lambda/genética , Clonación Molecular/métodos , Expresión Génica , Vectores Genéticos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Cinética , Datos de Secuencia Molecular , OligodesoxirribonucleótidosRESUMEN
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.
Asunto(s)
Fragmentos de Inmunoglobulinas/genética , Toxoide Tetánico/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Bacteriófago lambda/genética , Secuencia de Bases , Clonación Molecular/métodos , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Técnicas Genéticas , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
We have cloned the cDNA encoding human liver glycogen phosphorylase (glycogenosis type VI) from a fetal brain cDNA library. Liver(L) and muscle(M) phosphorylase cDNA probes were used to determine the relative abundance of mRNA encoding the L- and M-isozymes of phosphorylase in human fetal and adult tissues. The transcript encoding the M-isozyme is 3.4 kb; the L-isozyme transcript is 3.3 kb. Transcriptional expression of the L-isozyme in human and primate tissues was found to differ from the isozyme's reported tissue specificity in non-primate mammals. Furthermore, using degenerate oligonucleotide probes to two different coding regions of M-phosphorylase, a novel 4.1-kb transcript was demonstrated to be present in human fetal and adult brain.