Construction Of A Large Single-chain Phage Antibody Library

In recent years, the study of antibody-based drugs has developed rapidly. Now 18 antibody-based drugs have been approved for clinical use aboard and the development of therapeutic antibodies has been one of the hottest fields in the discovery of biological engineering drugs. The focus of therapeutic antibody development has been shifting to human antibodies because of their advantage of non-immunogenic and suitable for in vivo use. Large antibody library now is one of the major platforms for making human monoclonal antibodies. A diversified phage antibody library with a size of 10¹⁰ 10¹¹ can provide an efficient approach for preparation of human antibodies.The object of this work was to construct a large single-chain phage antibody library to provide a platform for human antibody preparation. Firstly, diverse VL and VH genes were amplified by RT-PCR from lymphocytes collected from normal adult peripheral blood and newborn cord blood. The V genes were spliced to form SCFV by overlap PCR and cloned into vector PDF, obtaining a primary library in which the VH were flanked by two non-homologous loxp site. Secondly, the library phagemid was transferred into cre-expressing bacteria BS1365 at high multiplicity of infection(MOI), through...