Construction,Preliminary Selection and Identification of Phage Display Library of Extracellular Region of Recombinant Anti-Human Endoglin scFv Antibody

Objective To construct the phage display library of extracellular region of anti-rhEndoglin scFv antibody.Methods Balb/c mice were immunized by the extracellular region of rhEndoglin and their mRNAs of the splenic cells were extracted.VH and VL were amplified by RT-PCR,which were ligated into scFv DNA through a linker,then the scFv DNA was recombined into pCANTAB5E vector. The recombinant plasmid was transformed to E.coli TG1.The construction of phage display library was finished after rescuing the transformed TG1 by Helper phage M13KO7.Five rounds of absorb-elute-enrich panning against rhEndoglin were done by phage-ELISA.The positive recombinant phage clones after phage-ELISA were used to extract the plasmids and the plasmids were sequenced.Results After panning,94 clones from the library were selected randomly and 37 positive clones were obtained.The result of sequencing showed that the scFv DNA was 738 bp,of which VH encoding 122 amino acid residues was 366 bp and VL encoding 109 amino acid residues was 327 bp.VH and VL were ligated by a peptide consisting of 15 amino acid residues in(Gly4Ser)3.ConclusionThe phage display library of the extracellular region of anti-rhEndoglin scFv was successfully constructed,from which a recombinant phage clone that could specifically bind with rhEndoglin was obtained by panning.