RESUMO
Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Assuntos
Animais , Humanos , Camundongos , Afinidade de Anticorpos , Células Cultivadas , Citidina Desaminase , Genética , Metabolismo , Células HEK293 , Região Variável de Imunoglobulina , Genética , Alergia e Imunologia , Mutação , Anticorpos de Cadeia Única , Química , Genética , Alergia e Imunologia , Hipermutação Somática de Imunoglobulina , Genética , Alergia e Imunologia , Fator de Necrose Tumoral alfa , Alergia e ImunologiaRESUMO
Fluorescence resonance energy transfer (FRET) is the energy transfer from an activated donor fluorophore to a receiving fluorophore. The efficiency of the energy transfer is the function of the distance between the two fluorophores, and is very sensitive to the distance. Its effective response distance is between 1~10 nm, thus it can be used to measure the distance between atoms or molecules. The feature of FRET is very useful in researches on conformational changes, interaction between macromolecules and signal transductions within live cells, and FRET has become a powerful tool in biomedical investigations. However, biological processes often involve interactions between more than two macromolecules, and FRET using two color fluorophores cannot meet the research demand. Recently, two research groups made breakthrough, establishing a novel FRET technique using three color fluorophores based on confocol microscopy and flow cytometry, respectively. The invention will significantly advance researches in biological and related fields.