| Summary: | 碩士 === 臺北醫學大學 === 醫學技術學系 === 94 === Severe acute respiratory syndrome (SARS) has been a newly emerged human disease. An effective method for rapid diagnosis or treatment of SARS should be developed. In this study, 10 truncated SARS-CoV spike proteins were fused with histidine and expressed in the E. coli BL-21 cells, The purity of these expressed proteins was verified using SDS-PAGE analysis. When incubated with sera from convalescent stage of 8 SARS-CoV-infected patients, it was observed that one protein located between amino acid residues 750 to 1000 was clearly recognized by 6 serum samples. These truncated spike proteins were then used to immunize domestic chickens for IgY antibody production. After 4 times of challenges, high titer of anti-spike protein antibodies were found in the sera and eggs of immunized chicken as demonstrated by the western blot and ELISA analysis. The total polyclonal IgY antibodies were partially purified and estimated to be 50-100 mg per egg. The specific binding ability of these IgY antibodies to SARS-CoV-infected cells was demonstrated by immunofluorescence assay. Furthermore, scFv (single-chain variable fragment) was expressed on the phage surface by cloning the heavy chain and light chain into pComb3H vector. After 4th panning procedure, 16 randomly selected scFv clones were examined for their binding to S proteins expressed by SARS-CoV infected cells. Sequence analysis showed that 44% clones with short linker and 43% clones with long linker contain identical heavy and light chain genes, indicating that particular clones were enriched through panning procedure. Of which, S8 and L5 scFv antibodies exhibited strong binding activity in immunofluorescence assay, which intensity is comparable to that demonstrated by polyclonal IgY antibodies. Using S8 scFv antibody, an antigenic epitope of S protein was mapped to the location spanning amino acid residues 456 to 650 using western blot. This result was further confirmed by ELISA method. In conclusion, we successfully generated scFv antibody with specific binding ability to SARS-CoV infected cells which may be helpful in the development of therapeutic agents in the future.
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