Construction Of Phage Display Library Of Fab Antibodies Repertoies And Identification Of Anti-hepatocellular Carcinoma Fabs

The emergence of repertorie cloning using phage antibody libraries has revolutionized the antibody engineering technology. It provides a powerful method to product human antibody. Fusion phage libraries expressing Fab antibodies were constructed from the lymphocytes with in vitro immunization with whole hepatocellular carcinoma cells. Two phage Fab clones were obtained, specially react with hepatocellular carcinoma cells but not with normal liver cells.Isolation and culture of lymphocytesThe lymphocytes were isolated from peripheral blood of thirteen hepatocellular carcinoma patients and tumor-draining lymph-node of a hepatocellular carcinoma patient. The lymphocytes were cultured with interleukin-2,pokeweed mitogen and lipopolysaccharide. Hepatocellular carcinoma cell line SMMC-7721 was dealed with stationary liquid or irradiated by ⁶⁰Co, then applied for immunizing the cultured lymphocytes in vitro. Lymphoblast-like cells occurred and colonies formed after immunization for 7 days.Construction of human combinatorial Fab librariesTotal RNA was isolated from cultured lymphocytes. Seven Fd fragment of the H chain (y) and 9 full-length light chain(6 X ,3 k) were amplified using degenerate oligonucleotide primers from total RNA with RT-PCR. A human combinatorial Fab library was constructed in the phagemid vector pComb3...