Effect of VL and VH consensus sequence-specific primers on the binding and expression of a mini-molecule antibody directed towards human gastric cancer

To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody (mAb)3H11. At first, 3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library a...

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Bibliographic Details
Published inChinese medical sciences journal Vol. 15; no. 3; p. 133
Main Authors Li, J, Wang, Y, Wang, Z, Dong, Z
Format Journal Article
LanguageEnglish
Published China 01.09.2000
Subjects
Animals
Antibodies, Monoclonal - genetics
Antibodies, Neoplasm - genetics
Binding Sites
Consensus Sequence
Escherichia coli - genetics
Escherichia coli - metabolism
Genes, Immunoglobulin
Humans
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - immunology
Immunoglobulin Fab Fragments - metabolism
Immunoglobulin Fc Fragments - genetics
Immunoglobulin Fragments - genetics
Immunoglobulin Fragments - immunology
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Light Chains - genetics
Immunoglobulin Variable Region - genetics
Mice
Mutagenesis, Site-Directed
Stomach Neoplasms - immunology
Stomach Neoplasms - pathology
Tumor Cells, Cultured
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Summary:To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody (mAb)3H11. At first, 3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was perfomed. Again the identification of positive clone was failed. Finally the N-terminal sequences of V regions were resumed to 3H11 original sequences by site-directed mutagenesis via PCR. Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected. Correction of either VL or VH N-terminal sequences could partially resume the antigen binding activity. Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
ISSN:1001-9294