Construction and Characterization of Single Chain Fv Phage Display Library Against Tumor Necrosis Factor α

This study was to construct single chain Fv (scFv) library against tumor necrosis factor α (TNF-α) by phage display technique and to characterize the anti-TNF-α scFv clones selected from the library.Total RNA was extracted from the splenocytes of the BALB/c mice immunized with recombinant human TNF-α (rhTNF-α).Complementary DNA fragments of variable heavy (VH ) and variable light (VL) chains of antibodies were prepared by RT-PCR and assembled into scFv with a flexible peptide linker (Gly3Ser)4.ScFv fragment was inserted into the phagemid vector pCANTAB 5E through Sfi Ⅰ/Not Ⅰ sites.The phagemides containing scFv cDNA were transformed into E.coli TG1 bacterial cells to generate scFv antibody library with a diversity of approximately 4.6×108.Three rounds of enrichment and panning were performed to select specific anti-TNF-α scFv from the library.The antigen binding activity was evaluated via ELISA assay.Sequencing analysis of one positive clone showed that the anti-TNF-α scFv was 774 bp,and coded 258 amino acids.The positive clone were transformed into E.coli HB2151 and induced with IPTG.The soluble scFv showed a molecular weight 28 kD analyzed by SDS-PAGE and Western blot.The affinity purified scFv exhibited the binding activity to rhTNF-α and could neutralize the cytolytic activity of rhTNF-α against L929 cells.In this work,a higher affinity anti-TNF-α scFv was obtained from scFv antibody library.These results laid the foundation for research of antibody for clinical immunization therapy.