Cloning of porcine scFv antibodies by phage display and expression in Escherichia coli.
Oligonucleotide primers were designed for recovery of Ig H, kappa and lambda transcripts from porcine splenic cDNA. The products were cloned and scFvs constructed in a phage display vector. E. coli HB2151 was transformed with the constructs and upon induction, scFvs of the predicted molecular weight could be detected in culture supernatants by Western blotting and immunoprecipitation with anti-pig IgG. Bacteriophage displaying a swine scFv were diluted into an excess of phage carrying a human anti-thyroglobulin scFv and then panned on plastic coated with anti-pig IgG. Rapid enrichment of phage carrying the porcine scFv through three rounds of selection demonstrated the successful display of an authentically folded pig Ig at the viral surface. The data demonstrate that phage display techniques can be applied successfully to porcine Igs and that expression of recombinant pig scFvs in bacteria can be achieved. These techniques offer the opportunity to generate authentic porcine monoclonal reagents for basic and applied studies.
Li
Fuqiang
F
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Aitken
Robert
R
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D005803:Genes, Immunoglobulin / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D013552:Swine / Q000235:genetics / Q000276:immunology*
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger; D011994:Recombinant Proteins