Functional Expression of NS2 & NS4 of Dengue Virus 2 and Mechanism Study of Tetracycline-Derivatives as Dengue Virus Inhibitors

• Main Authors: Chang, Ching-Yun, 張瀞云
• Other Authors: Yang, Yun-Liang
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/35427608342711438019

碩士 === 國立交通大學 === 生物科技系所 === 97 === Dengue viral infections have become a global health issue in tropical and subtropical regions. There were outbreaks reported in more than 100 countries and about 2.5 billion people lived under the threats. Therefore, the developments of vaccines, treatments, or prevention measures for dengue viral infections are a pressing issue. Dengue virus encodes 10 proteins in a single open reading frame. Among them, NS2A, NS2B, NS4A and NS4B are small non-structural proteins exhibiting hydrophobicity profiles. NS2B has been suggested to involve in protease activity while specific biological functions for NS2A, NS4A and NS4B have not been identified. With the interest to study the structure and function of these four proteins, I attempted to clone, express, and functional assay of these four genes. Their proteins were expressed as EGFP (enhanced green fluorescence protein) fusion proteins in mammalian cells. The stable cell lines of NS2A and NS2B were successfully selected and confirmed by Western blot analysis. The results showed that NS2A-EGFP effected a 44% reduction of plaque numbers compared to the negative controls, BHK-21 cells (P <0.05). Previously, our lab has identified certain tetracycline derivatives as potential inhibitors to Dengue virus, by docking into the β-OG pocket on the DV2 E protein. Hence, I set out to test various tetracycline compounds for their effect on dengue viral propagation of DV2 PL046 strain and DV3 H87 strain by plaque formation assay. All the tetracycline derivatives tested inhibited the plaque formations on DV2 and DV3 in cell culture systems, especially of doxycycline and chlortetracycline. And in comparison, the inhibitory effect on DV2 is better than that on DV3.