RESUMEN
We selected useful antibody fragments against rabies virus from a human single chain variable fragment (scFv) gene library using ribosome display technique. The recombinant rabies virus glycoprotein (RVGp) was used as an antigen to isolate specific scFvs. After five rounds of selection, the analysis demonstrated that scFv-ribosome-mRNA complexes were specifically selected against RVGp. Sequence analysis showed that mutations were introduced at random by PCR among the rounds of selection and variants with high affinity were isolated. The obtained scFvs with high affinity could recognize RVGp specifically and showed binding activity to rabies virus. These scFvs were potential for inclusion in a combination of several human monoclonal antibodies (MAbs) aimed for application in rabies post-exposure prophylaxis. Ribosome display technology is a robust tool for rapid isolation of human antibody fragments, and has exceptional strength in affinity maturation and molecular evolution in vitro.
Asunto(s)
Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos , Biblioteca de Genes , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Virus de la Rabia/inmunología , Ribosomas/metabolismo , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Biotecnología/métodos , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Selección Genética , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
AIM: To prepare distinct human McAbs to TNF-alpha with high neutralizing potency using ribosome display technology. METHODS: The immunoglobulin heavy and light chain variable (VH, VL) genes were prepared from the peripheral blood lymphocytes in three arthritis patients by PCR. The genes encoding VH/K fragments were prepared by randomly combining VH and VL genes by SOE PCR. TNF-alpha binding fragments were selected over three cycles of ribosome display and the selected VH/Ks genes were cloned into pET22b(+)/BL21(DE3), from which soluble VH/K fragments were prepared. The expressed products of selected clones were analyzed by ELISA. Then the positive clones were characterized. RESULTS: A human VH/K gene library with 6.7x10(12) numbers used for ribosome display was constructed. Among the 180 selected clones, two clones TRB21 and TRB409 exhibiting the highest ELISA signals were isolated. The analysis of the sequence of TRB21 and TRB409 showed that they were new human immunoglobulin V genes to TNF-alpha and they recognize TNF-alpha specifically and antagonize the cytolytic effect of TNF-alpha on 1929 cell. CONCLUSION: The selected VH/Ks to TNF-alpha will be useful for constructing engineering antibodies with high affinity against arthritis. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.