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[Construction and expression of ScFv from anti-human gastric cancer McAb 3H11]
Objective:
To construct single chain antibody (ScFv) from anti-gastric cancer McAb 3H11.
Methods:
Phage display technology was used to construct ScFv library from 3H11 hybridoma cells. Site-directed mutagenesis was used to resume the N-terminal sequences of 3H11 ScFv.
Results:
The library was screened against human gastric cancer cells MGC803 but no positive clone was obtained. Then, a random mutated ScFv library was constructed by error-prone PCR method, and panning selection was performed. Again the identification of positive clone was failed. Finally the N-terminal sequences of ScFv variable region was resumed to 3H11 original sequences by site-directed mutagenesis via PCR, and the expressed ScFv in bacterial culture supernatant showed binding activity to gastric cancer cells.
Conclusion:
The N-terminal of the variable region introduced by PCR primers may seriously affect binding activity of the antibody.