Construction And Screening Of A Phage-displayed ScFv Antibody Library Against Human Gastric Cancers

To construct a phage-displayed library of human single-chain antibodies and identify specific antibodies associated with human gastric cancers.Methods and results:The cDNA library of antibody variable regions was cloned from peripheral blood lymphocytes of patients with gastric cancers by RT-PCR.The heavy-chain(V_H) and light-chain variable region genes(V_L) of the antibodies were amplified by PCR.The V genes were assembled to form scFv by overlapping PCR and cloned into phagemid pCANTAB-5E,then transformed into E.coli TG1.The scFv gene library was rescued by M13KO7 helper phage.To identify specific antibodies against gastric cancers,we developed a strategy to remove antibodies cross-reacting with common antigens on normal cells by panning on normal human gastric mucosal epithelial cells.After five rounds of subtractive panning,positive clones were confirmed by ELISA to determine the specificity of phage antibodies.We finally identified 5 clones that bound selectively to MGC-803 cells.The corresponding scFv sequences were deduced by DNA sequencing and the tertiary structures were predicted using UCSF Chimera. Summery:We successfully constructed a human phage single-chain antibody library and isolated specific antibodies reacting with gastric cancer cells.The diversity of the library is good.It also shows that the positive clones have excellent specificity. The scFv fragment may be further developed and applied to clinical diagnosis and therapy.