Solubility Enhancement and Purification of VP1 in EV71

• Main Authors: Tian-you Hsiao, 蕭天佑
• Other Authors: Cheng-I Lee
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/5n3xwc

碩士 === 國立中正大學 === 生物醫學研究所 === 104 === Enterovirus 71 (EV71) is a subgroup of human enterovirus (HEVs), major infectious agents of hand, foot and mouth disease (HFMD), that can potentially cause severe neurological diseases resulting children death. Thus, the occurrence of EV71 infection has been monitored every year by Ministry of Health and Welfare in Taiwan. HEVs are spread through the excreta-oral route and through secretions such as saliva and nasal mucus. The components of mature virion in EV71 have components VP1, VP2, VP3 and VP4 to assemble into an icosahedral shell. Previous studies have showed that VP1 of EV71 can bind scavenger receptor B, member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) to infect human cells. VP1 of enterovirus 71 is a key protein in infectious pathway and participates in RNA release. There are many loops in VP1 that can be the targets of antibodies for virus neutralization. Therefore, it is essential to study the biochemical role of native VP1. To obtain VP1 protein for further study on EV71, we need to have a large scale expression and purification of VP1. Primarily, we used E. coli system transformed with pET21a-VP1 to express VP1, but the proteins were expressed in inclusion body. Purification of proteins in inclusion body requires denaturation which becomes an obstacle in further functional study. Lately, we have a construct pET32-trx-VP1 that brings the expressed VP1 into soluble form. Based on the soluble VP1, we have carried out a serious of protein purification for highly pure VP1. The final purified product will be applied in antibody generation and further biochemical studies on VP1.