Construction, Expression And Activity Identification Of Anti-HER2 ScFv/Ck/tP Fusion Proteins

Objective:To construct genes of anti-HER2 ScFvCk/tP fusion proteins containing Furin-cleavable peptide and muclear localization signal, and to study their binding activities and internalization functions after they were expressed and purified in E coli.Method:A pair of oligonucleotide primers was designed and used to amplify the ScFv e23sFv gene. After Ck was ligated with it as ScFv/Ck, the synthesized coding sequence containing Furin-cleavable peptide, G4S, tP, G4S and NLS was added in the 3'terminus, then the recombined fusion protein gene ScFv/Ck/FCS/G4S/tP/G4S/NLS was obtained, based on this gene, the ScFv/Ck/FCS/G4S/tP,ScFv/Ck/tP/G4S/NLS,ScFv/Ck/tP,ScFv/Ck genes were also constructed, they were cloned into expression vector pET28a and pET32a. After induced in E.coli BL21 (DE3) by IPTG, expressed proteins were detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. The antigen-binding activity of the fusion proteins were analyzed by cellular ELISA, the DNA binding ability were verified by Gel shift assay, and the internalization function was confirmed by indirect immunofluorescence staining.Result:Restriction endonuclease digestion analysis and DNA sequencing proved that the amplified ScFv sequence was correct, and the expression plasmids of anti-HER2 ScFvCk/tP fusion proteins containing Furin-cleavable peptide and NLS were successfully constructed. SDS-PAGE and Western blot analysis showed that the fusion proteins were successfully expressed and purified after induced in E.coli BL21 (DE3) by IPTG. Cellular ELISA confirmed that all the fusion proteins maintained antigen binding activities. Gel shift assay assured that the fusion proteins containing tP had DNA binding activity. Indirect immunofluorescence staining revealed that they were able to internalize into HER2 positive cells but not into HER2 negative cells. The fusion protein containing Furin-cleavable peptide and NLS had much better internalization function.Conclution:The fusion proteins maintained the binding activities to HER2 and obtained DNA binding activities after fused with Ck, Furin-cleavable peptide, tP and NLS. They were able to internalize into HER2 positive cells specifically. The fusion protein which containing Furin-cleavable peptide and NLS both had better internalization effects. Our present studies may provide a theoretical foundation for the application of this ScFv in targeted delivery of DNA or siRNA.