Selection of anti-cancer-associated gene single-chain variable fragments derived from gastric cancer patients using ribosome display

• Published in: Molecular medicine reports Vol. 8; no. 2; pp. 631 - 637
• Main Authors: XIN, LIN, CAO, JIAQING, LIU, CHUAN, ZENG, FEI, CHENG, HUA, HU, XIAOYUN, ZHU, PEIQIAN, SHAO, JIANGHUA
• Format: Journal Article
• Language: English
• Published: Greece D.A. Spandidos 01.08.2013; Spandidos Publications UK Ltd
• Subjects: Affinity; Amino Acid Sequence; Antibodies; antibody engineering; Antibody Specificity - genetics; Antibody Specificity - immunology; Antigen-antibody complexes; Antigens; Antigens, Neoplasm - immunology; Base Sequence; cancer-associated gene; Cell Surface Display Techniques; Cloning; Cold; Construction; Deoxyribonucleic acid; DNA; E coli; Enzyme-linked immunosorbent assay; Gastric cancer; Gene pool; Humans; Immunoglobulins; Lymphocytes; Molecular Sequence Data; Patients; Peripheral blood; Polymerase chain reaction; Reverse transcription; ribosome display; Ribosomes; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; single-chain variable fragment; Splicing; Stomach Neoplasms - genetics; Stomach Neoplasms - immunology
• ISSN: 1791-2997; 1791-3004
• DOI: 10.3892/mmr.2013.1502

The aim of this study was to construct a human single-chain variable fragment (scFv) gene library for gastric cancer, from which human anti-cancer-associated gene (CAGE) scFvs are selected. Human lymphocytes were isolated from the peripheral blood of 10 gastric cancer patients and whole human heavy and light chain genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR). VH and VL were rearranged randomly by splicing by overlap extension PCR. The ribosome complexes were enriched against the recombinant CAGE protein conjugated to magnetic beads. scFv antibodies were evaluated by western blot analysis, and affinity constants in a solution of antigen-antibody complexes were determined by enzyme-linked immunosorbent assay. An scFv library was constructed using the peripheral blood lymphocytes. The expressed scFv proteins from the ternary ribosome complexes were analyzed by western blot analysis and the affinity [equilibrium dissociation constant (KD)] of scFv for CAGE was determined to be 7.6×10−8 M. The ribosome display technique is efficient for selecting a fully human antibody fragment from a patient-derived gene pool.