Gene construction,protein purification and activity evaluation of scFv anti-CD5

Purpose To construct a genetic engineering E.coli strain with high expression level of scFv anti-CD5 and to lay the foundation for tumor immunotherapy using scFv anti-CD5 as a component.Methods VH and VL fragment of H65(an anti-CD5 mAb)is acquired from Genbank,and then joined with a linker coding sequence,and the gene via overlapping PCR is constructed.The gene is digested and inserted into pET22b(+) to form a recombinant plasmid.The E.coli BL21(DE3) bear the plasmid with right sequence is induced with IPTG and the product is purified with Ni-NTA column,renatured,and subjected to activity analysis via FCM and Western blotting.Results The recombinant plasmid and the engineering E.coli strain with high expression level of scFv anti-CD5 were acquired.The expression products were able to recognize CD5 antigen after being renatured.Conclusion The acquisition of engineering E.coli strain with high expression level of scFv anti-CD5 would contribute to further application of scFv anti-CD5.