Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin

• Published in: Journal of biochemistry, molecular biology and biophysics Vol. 6; no. 1; p. 45
• Main Authors: Nathan, Sheila, Li, Hongbin, Mohamed, Rahmah, Embi, Noor
• Format: Journal Article
• Language: English
• Published: England 01.02.2002
• Subjects: Amino Acid Sequence; Animals; Antibodies, Bacterial - genetics; Antibodies, Bacterial - immunology; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Base Sequence; Burkholderia pseudomallei - immunology; Burkholderia pseudomallei - pathogenicity; Cell Transformation, Viral; Cloning, Molecular; Enzyme-Linked Immunosorbent Assay; Exotoxins - immunology; Hybridomas; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Light Chains - genetics; Immunoglobulin Variable Region - genetics; Mice; Molecular Sequence Data; Peptide Library; Recombinant Fusion Proteins - immunology; Sequence Analysis, Protein
• ISSN: 1025-8140
• DOI: 10.1080/10258140290010232

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.