Screening scFv Specific to Vcam-1 by Phage Display Library and Its Activity Evaluation / 华中科技大学学报(医学版)

ABSTRACT

Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones were transferred into E.coli ,and the clone with the highest expression was regarded as the final requisite one.Competent cells were infected by the final requisite clone and scFv was expressed.After purification ,the activity of scFv was tested by ELISA and its affinity was evaluated.Results Molecular weight of Vcam‐1 antigen protein was 85-90 kD.Positive clones were screened out by taking Vcam‐1 protein as the antigen ,and 9 high titer clones were obtained by single phage ELISA.Gene sequencing of these clones was carried out and 3 sequences were obtained ,1 of which got the highest expression.Molecular weight of the expressed scFv was about 30 kD.The scFv got high affinity to Vcam‐1 antigen according to ELISA ,in spite of its lower activity than full‐length monoclonal antibody.Conclusion scFv antibody specific to Vcam‐1 was successfully obtained from phage display librar‐y ,which laid the foundation of subsequent in vivo diagnosis and therapy.