| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 92 === Abstract
A single-chain variable fragment (ScFv) is the combination of heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, connected by a polypeptide linker. Due to the small size and the simplicity in engineering, yet with specificity to antigen, ScFv is a promising alternative to monoclonal antibody in both clinic and research applications. Conventionally, specific ScFv is isolated from a ScFv library by screening against an interested antigen using phage-display technology. The aim of the study is to generate ScFv against either recombinant Schistosoma japonicum glutathione-S-transferase (GST) or recombinant SARS-CoV nucleocapsid protein. Mice were immunized with either GST or nucleocapsid protein. The VH and VL cDNA fragments were generated by RT-PCR using mRNA isolated from spleens of immunized mice. The ScFv libraries were constructed by overlapping PCR, with VH and VL cDNA as templates. Potential ScFv clones were isolated from the libraries by phage-display screening. To further confirm the specificity of ScFv to their antigens, the ScFv candidates were purified by immobilized metal affinity chromatography and then were assayed. We showed that the purified ScFv was able to bind either GST or nucleocapsid protein, respectively by ELISA. In addition, recombinant nucleocapsid protein could be detected by ScFv using nucleocapsid protein-capture ELISA. These data indicated that the ScFv could specifically recognize its antigens. The ScFv might be applied to the detection of GST and nucleocapsid protein.
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