Gene cloning,expression and binding activity of anti-CD86 scFv

Objective To construct and express the single chain variable fragment(scFv) against human CD86 in CHO cells,and observe its biological functions of binding with antigen.Methods The VH and VL genes were cloned by RT-PCR from a murine hybridoma cell line 1D1,which produced the monoclonal antibody(mAb) against human CD86.The CD86-scFv gene was integrated into eukaryotic expression vector to construct pIREST2-EGFP/scFv.CHO cells were transfected by pIREST2-EGFP/scFv plasmid with LipofectamineTM 2000 and then were selected by G418.We used IMAC affinity chromatography to purify CD86-scFv and quantified its concentration by BCA method.Then the identification of CD86-scFv binding to membrane CD86 was performed through competitive inhibition assay.In addition,the growth inhibition effect of CD86-scFv on Raji cells was detected via MTT assay.Results One cell line stably expressing CD86-scFv was obtained.The CD86-scFv could identify CD86 molecules on L929-CD86,Raji and Daudi cells,and the positive rates were 67.0%,72.3% and 80.5%,respectively.CD86-scFv showed the competitive binding to murine parent antibody 1D1.Moreover,CD86-scFv inhibited the growth of Raji cells and the inhibition rate was 28.3% 72 h after 20 μg/mL CD86-scFv was added into Raji cells.Conclusion CD86-scFv has been successfully expressed in CHO cells(named SA-IV) and the antibody shows a good biological function of recognizing CD86 and inhibiting the growth of Raji cells.