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[Construction and expression of human anti-HER2 scFv-9R fusion protein and identification of its activity]
Kuo Jiang1, Rui Chen, Li-feng Wang
1Orthopedics Oncology Institute of Chinese PLA, Tangdu Hospital, Xi'an, China. jiangkuo520@gmail.com
Aim:
To construct, express and purify a human fusion protein, which is composed of a single-chain antibody fragment scFv that recognizes HER2 protein and an oligo-9-arginine, and to analyze the binding activity of the expressed fusion protein.
Methods:
Pairs of oligonucleotide primers were designed and used to amplify the scFv-9R. The fusion protein gene scFv-9R was then cloned into expression vector pQE30 and expressed in E.coli M15.Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. Then, the purified protein was refolded by dialysis and concentrated by ultrafiltration. The antigen-binding activity of the scFv-9R fusion protein was confirmed by ELISA, and the siRNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA).
Results:
HER2 scFv-9R encoding sequence was correctly cloned into the expression vector. The recombinant protein was insolubly expressed in E.coli M15 induced by IPTG. ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had siRNA binding activity.
Conclusion:
The scFv-9R fusion protein can specially bind with both HER2 antigen and siRNA.
