| Summary: | 碩士 === 中興大學 === 生物科技學研究所 === 95 === Natural polymers and virus capable of self-replicating and assembly draw many scientists into the development and application of these materials in nanotechnology. Many applications and material development are based on the manipulation of various peptides on the surface of virus particle. Turnip yellow mosaic virus (TYMV) is an icosahedral virus, 28 nm in diameter. It comprises a positive-stranded RNA genome and 180 copies of capsid protein. In a previously study, a mutant virus (TYMV/Cys) which displays one cysteine residue at the C terminus of viral coat protein was constructed showed a similar infectivity to that of wild type TYMV. TYMV/Cys provides 110 and 400 of thiol and amine groups, respectively, on the surface which could conjugate with different kinds of fluorescent markers. Since the C terminus of coat protein on the virus surface is predicted to be in a cleft, the location may be sterically hindered for further applications. A new construct, TYMV/A6C, containing 6 alanine residues with a C-terminal cysteine is predicted to extend the thiol group from the cleft. Characterization of TYMV/A6C indicates that it could provide about 90 and 400 of thiol and amine groups, respectively, on the viral surface. TYMV/A6C was further used to be conjugated with 1.4 nm-diameter nano-gold particles through the thiol groups and the labeling was observed under transmission electron microscope. Purified antibody and fluorescein were conjugated to the thiol and amine groups, respectively, on TYMV/A6C and subsequently purified with an ion-exchange column. This fluorescently labeled viral particle conjugated with antigen specific antibody was tested in a fluorescent immumosorbent assay and compared to the conventialthe sensitivity of conventional ELISA (enzyme-link immunosorbent assay) system.
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