Constructing And Screenning A Natural Phage Antibody Library Of Human Anaplastic Thyroid Carcinoma

Objective:The objective of this study was to identify and construct a human natural phage single-chain antibody (scFv) library of human anaplastic thyroid carcinoma (ATC) using phage display technology.Methods:Total RNA was extracted from lymphatic tissue near an ATC and used to amplify fragments consisting of a variable heavy chain (VH) and a variable light chain (VL) with reverse transcription polymerase chain reaction (PCR) and join with their linkers. After purification, VH and VL were used to produce the scFv fragments using splicing-overlap-extension (SOE) PCR, and then loading on the restricted enzyme cutting sites sfi I and Not I. The scFv gene was cloned in the pCANTAB-5E plasmid, and the recombinant phagemids were transformed to susceptible Escherichia coli TG1.Panning against ARO cell line was performed for four rounds and the antibody library was identified by ELSIA.Result:After infection by the helper phage M13K07, the human ATC phage antibody library was successfμlly constructed, the scFv gene were approximately 750 bp. And the conversion efficiency measured by the pUC19 standard plasmid was 108 CFU/μg, and the positive insert ratio was 86.4%(19/22). Moreover, the antibody was enriched with the increase of panning rounds, and the fourth one harvest yielded 77 times as much as that of first one. The ELISA resμlts showed that the soluble scFv was highly active and coμld bind ARO cells, but not TT cells.Conclusion:The human ATC scFv antibodies gene library was successfμlly constructed and the selected scFv gene can specifically bind to human ARO cell line. It coμld provide the experimental basis for further radioimmuno imaging of the phage single-chain antibody with cell-specific ATC.