| Summary: | 碩士 === 國立宜蘭大學 === 生物技術與動物科學系生物技術碩士班 === 106 === Human norovirus is one of the major pathogens causing acute gastroenteritis. Eating raw aquatic animals, such as shellfish and oyster, is the main route of transmission. Taiwan is an important hub for import/export of aquatic products, and rapid detection of pathogenic contamination is associated with the health maintenance of citizens in Taiwan and economic benefits of aquatic products. In Taiwan, acute gastroenteritis is mainly caused by GI or GII norovirus. At present, Taiwan has not developed antibodies for viral testing. This study intends to develop specific antibodies for GI.9 and GII.17, which are common in Taiwan, for the investigation of infection mechanism of norovirus. Rapid testing reagent for norovirus can be developed in the future and applied to border testing of aquatic foods to be imported or exported to protect the food safety of citizens in Taiwan.
Based on a report by the Taiwan Food and Drug Administration (TFDA), we have already obtained the norovirus RNA isolated from the oysters and have confirmed that they are type GI.9 and GII.17 after DNA sequencing. The GI.9-VP1-P-domain gene (987 bp)、GII.17-VP1-S-domain gene (597 bp) and GII.17-VP1-P-domain gene (1005 bp) from the two types of norovirus have been subcloned into the pET23a(+) prokaryotic expression vector. After transformation into E.coli BL21 and IPTG induction, these recombinant proteins were purified by Ni-affinity column. The GI.9-VP1-P-domain-His with a predicted molecular mass of 38.5 kDa, GII.17-VP1-S-domain-His is 25.9 kDa and GII.17-VP1-P-domain-His is 40 kDa and will subsequently serve as antigens to immunize mice and rabbit for antibody preparation. These three antigens all induce high antibody titer in mice and rabbit. Three mouse monoclonal antibodies against GI.9-VP1-P domain, two mouse monoclonal antibodies against GII.17-VP1-S-domain, and seven mouse monoclonal antibodies against GII.17-VP1-P-domain were established. Finally, we also established a SW480-FUT2 cell line stably expressing FUT2 may serve as a valuable cell model for norovirus infection study.
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