| Summary: | 碩士 === 中臺科技大學 === 食品科技研究所 === 97 === Abstract
VP2 is a major structural protein of Infectious bursal disease virus (IBDV), which contains only two Cysteine residues, Cys-99 and Cys-197, located at SD domain and PA’ domain, respectively. In this study, VP2 was engineered by site-directed mutagenesis to investigate the role of Cysteines on the formation of VP2 subviral particle (SVP). Mutants VP2-C99A, VP2-C197A, VP2-C197T, VP2-C197S and VP2-C99.197A were expressed in E. coli and VP2-C99A and VP2-C197A were also expressed in insect cell/ baculovirus system. VP2-C99A expressed in both systems remains its ability of SVP formation and can be purified by immobilized metal-ion affinity chromatography (IMAC), with a particle size of 20-25 nm. The other mutants expressed in E. coli lost their affinity to Ni-NTA resin, indicating that they do not exist in SVP form. Most of the insect cell-derived VP2-C197A was present in the flow-through and pH 7.8 binding buffer eluant, suggesting that they have lost the absorption ability with Ni-NTA. The small amount of VP2-C197A purified by IMAC exhibits swelling and incomplete particle form observed under electron microscope (EM). Our results indicate that Cys-197 is an essential and important site for the self-assembly of VP2 into subviral particle.
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