[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.


CONCLUSIONS
The N-terminal of the variable region introduced by PCR primers may seriously affect binding activity of the antibody.
School of Oncology, Beijing Medical University.
J
J
Li
Y
Y
Wang
Q
Q
Li
D000911:Antibodies, Monoclonal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D013274:Stomach Neoplasms / Q000276:immunology* / Q000473:pathology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library