Construction and selection of human anti-idiotypic antibody single chain variable fragments or CDR3 fragments of nasopharyngeal carcinoma.

摘要

Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). Detection showed that of 10 NPC patients, 8 patients' B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of VL genes were obtained by RT-PCR amplification and then connected with (Gly4Ser)3 linker to form 14 types of scFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into E. coli MC 1061. Phage anti-idiotypic antibody library with 1.5 x 10(8) clones was obtained. After four rounds of panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. 5 clones (D83, E92, G22, I50, I54), which might display beta type Ab2 scFv, were selected by binding inhibition test. These 5 phage anti-idiotypic antibodies were further analyzed by DNA sequencing. The VDJ regions of G22, I50, I54 belonged to VH4-39-D4-11-JH3-linker-V1-19-JL2, VH4-4-D4-11-JH6 and VH4-31-D4-11-JH6, respectively. E92 had the same VDJ regions with G22; D83 had the same VDJ regions with 150. So, a strategy for preparing and selecting beta type Ab2 scFv or CDR by means of immunization in vitro, EBV transformation and phage display technique is feasible, which paves a way for preparing cancer vaccine using beta type Ab2 scFv.