Study of the different promoters and virus internal ribosome entry site (IRESs) activity in the Koi fin cells (KFC) , mammalian cells and insect cells

• Main Authors: Chun-Chung Chen, 陳俊中
• Other Authors: Tzong-Yuan Wu
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/11982799359310440244

碩士 === 中原大學 === 生物科技研究所 === 97 === Protein expression systems are important tools in modern biotechnology. Many expression vectors provided for the different expression system to produce protein have been developed, except the fish cell systems. The promoter is one of the elements for the expression vectors that regulate gene expression on transcription level and have specific for different type cells. To finding the promoter that is optimal in the different cells, CMV, Krt4, Hsp70, beta-actin and Cmlc2 promoter was test in Koi Fin cells (KFC), mammalian cells and insect cells. In addition, to produce more than two of proteins in the same vector, dicistronic vector was used for this study. A IRES element was constrstruct into the expression vector to produce the second protein that was translated by a mechanism of cap-independent translation from the same transcript. The IRESs: EV71, EMCV and RhPV IRES were tested in the KFC cells and mammalian cells. The results demonstrate the CMV promoter have strong activity in mammalian cells, especially for the CHO cells and its activity is more than others about fifteen times. Nevertheless, all of the promoters were weak in the KFC by the transient transfection assay. The Hsp70 promoter seemed to be better than other promoters except compare with CMV promoter either in KFC or mammalian cells. In addition, the EV71 IRES activity was stronger than RhPV and EMCV IRES activity in mammalian cells although RhPV IRES as well as EV71 can function well in the KFC.