Expression and detection of ScFvB9 and its mutant in recombinant phage antibody system.
Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL). In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E. coli. The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E. coli. The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS.
Yang
E J
EJ
Dept. of Food and Nutrition, Chosun University, Kwangju 501-759, Korea.
Kwak
J W
JW
Chang
H C
HC
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv