Molecular Characterization of the Human Polyomavirus JC Virus Major Capsid Protein Expressed in Insect Cells and Investigation of its Capsid Stability

• Main Authors: Fung, Chiung-Yau, 方瓊瑤
• Other Authors: Deching Chang
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/50603924307033412911

碩士 === 中山醫學院 === 生物化學研究所 === 84 === The gene of the major capsid protein VP1 of human polyomavirus JC virus has been cloned into baculovirus and expressed in insect cells. The VP1 protein was able to self-assemble into a capsid structure in insect cells. The VP1 protein was purified by CsCl density gradient centrifugation to near homogeneity.The density of this purified capsid was 1.29 g/ml. Electron microscopy demostratedthat the VP1 protein was expressed in the cytoplasm and then transported into the nucleus. Two-dimensional gel analysis revealed that the VP1 proteins were composedof six distinct species. EDTA and DTT together were able to disrupt the capsid into capsomeres. Zn and Cd couldreassembled capsomeres into capsid. In additions the reassembled capsid could bedisrupted into capsomerew by EDTA alone. These findings indicated that the divalentcations played a major role in assembling the capsid structure and the disulphidebond protected the cations from chelation. Capsid appeared to be more stable inneutral or alkaline conditions (pH 6.5~ 10.5). Futhermore, capsid was found to be more stable at 37C and 42C, but not at above 65C.