A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage.
Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.
Lee
Yu-Ching
YC
Graduate Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan.
Leu
Sy-Jye C
SJ
Hung
Han-Chang
HC
Wu
Hsueh-Hsia
HH
Huang
I-Jen
IJ
Hsieh
Wen-Shyang
WS
Chiu
Wen-Ta
WT
Hsieh
Ming-Song
MS
Cheng
Tsui-Fen
TF
Yang
Yi-Yuan
YY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007136:Immunoglobulins / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D045169:Severe Acute Respiratory Syndrome / Q000276:immunology*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
C016512:IgY; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D019151:Peptide Library; D012367:RNA, Viral; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins