| Summary: | 博士 === 臺北醫學大學 === 藥學研究所 === 95 === The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein (S) were isolated and characterized. We expressed ten truncated S protein gene fragments in Escherichia coli cells. Following the immunization of chickens with these recombinant S proteins, two single-chain variable fragment (scFv) antibody libraries were established with short and long linkers to contain 5 × 107 and 9 × 106 transformants, respectively. After four rounds of panning selection, the scFv-expressing phages and scFv antibody molecules of individual clones were used to identify antigenic epitopes on S protein. In a comparison of nucleotide sequences with the chicken germline gene, five reactive scFv-expressing phage clones differ from 31% to 62% in complementarity determining regions. Of which, one dominant phage (4S1) had strong binding to a fragment located between amino acid residues 456 to 650 of S protein. This particular phage also showed significant binding to SARS-CoV-infected Vero E6 cells. When the soluble scFv antibodies was applied for antigenic mapping, two clones (Ssc35 and Lsc18) reacted strongly with SARS-CoV-infected Vero cells, and that significant sequence variation of these two clones in the complementarity-determining regions was also seen as that in scFv-expressing phage. In addition, the scFv expression was visualized by Coomassie blue staining, and detected by Western blot analysis. However, it is notable that those two specific scFv antibodies recognized the same region of the S protein spanning amino acid residues 750 to 1000, which differs from that identified by scFv-expressing S1 clone. The reason for this discrepant epitopic mapping by scFv displayed on phage surface and soluble scFv antibodies is presently unknown and needs further investigation. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model to produce high-affinity binders against the SARS-CoV S protein and this knowledge can be applied in clinical or academic research.
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