Construction of anti-SSA phage single-chain Fv antibody library

Objective To construct anti SSA phage single chain Fv antibody library by phage display technology.Methods RNA was isolated from the peripheral blood mononclear cells (PBMC) of patients with anti SSA antibody and reverse transcribed to cDNA,amplified VH and Vκ and linked them together with a linker segment by overlapping PCR to form VH linker Vκ (scFv gene),cut the scFv gene and phagmid vector pHEN 2 by Sfi Ⅰ and Not Ⅰ,ligated them and electrically transformed to TG1.Twelve clones were randomly selected to amplify the insertion fragment by PCR.The TG1 containing pHEN2 scFv was infected by helper phage VCS M13 to produce scFv phage antibody.The anti SSA activity of the phage antibody was tested by ELISA with the plate coated by purified SSA antigen.Results The fragment of VH and Vκ were about 300 to 400 bp.They were linked successfully by overlapping PCR in vitro to form scFv of about 800 bp.After cloning scFv to phagmid pHEN 2 and transforming pHEN 2 scFv to the TG1,3 0×10 7 clones formed.The insertion was amplified in 11 of the 12 randomly selected clones by PCR.After VCS M13 infection,1 2×10 14 pfu/ml scFv phage antibody were produced.In the anti SSA ELISA,the A produced by scFv phage antibody was 2 0 to 2 2 times of that produced by VCS M13.Conclusion The scFv phage antibody library contains about 3 0×10 7 individual clones,and the insertion efficiency is about 91%.This phage antibody library contains scFv antibody with the anti SSA activity.It can be used for the selection of monoclonal anti SSA phage antibody.