Generation and characterization of chicken antibody fragments against SARS-CoV 3a & 3b protein by phage display

• Main Authors: Che-Kun Cheng, 鄭徹堃
• Other Authors: 呂思潔
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/03780196147357888790

碩士 === 臺北醫學大學 === 醫學科學研究所 === 96 === 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. 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. The expression of SARS-CoV 3a alone can up-regulate the expression of fibrinogen in lung cells. A recent study showed that 3a is a novel coronavirus structural protein suggesting that 3a may be important to SARS-CoV. In this study, truncated SARS-CoV 3a and 3b proteins were fused with histidine and expressed in the E. coli BL-21 cells. The purities of these expressed proteins were verified using SDS-PAGE. The protein molecular weights are 34.2kDa and 28.1kDa. The Leghorn chickens were immunized by intramuscular injection with purified SARS-CoV 3a or 3b fusion protein. After 4 times of challenges, high titer of anti-3a and anti-3b protein antibodies were found in the eggs of immunized chicken as demonstrated by the Western blot and Enzyme-linked immunosorbent assay. The specific binding ability of these IgY antibodies to SARS-CoV-infected cells were 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 pComb3X vector and then infected the XL1-Blue E.coli with VCSM13 phage. A phage displaying scFv library was also established from spleen B cells of immunized chicken. Generation of scFv antibody with specific binding ability to 3a and 3b protein and SARS-CoV infected cells is still under investigation which may be helpful in the development of therapeutic agents in the future.