| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === Dengue virus is one of the most important pathogens in the tropical and subtropical areas. Dengue virus belongs to the family Flaviviridae, and has four serotypes (DV1 to DV4). The envelope of dengue virus contains the precursor membrane and envelope proteins (prM/E) encoded by the virus. The E protein belongs to type I membrane protein and has a stem-anchor region at its C-terminal. The anchor region contains two transmembrane segments, which anchors the E protein to the membrane. Previous studies showed that if the entire prM and E protein of tick-born encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) is expressed, the production of extracellular virus-like particles (EPs) is efficient. However, abundant EPs production of DV2 can be achieved, only if the stem-anchor region (C-terminal 20%) of the DV2 E protein is replaced by that of JEV. In the presence of authentic DV2 E protein or if only the anchor region (C-terminal 10%) of DV2 E protein is replaced by that of JEV, EPs production is not efficient. Therefore, the stem-anchor region of JEV sequence contains some enhancing elements for large production of EPs.
The main purepose of this study is to investigate the domains or critical amino acids in the stem-anchor region of the DV2 E protein that contribute to the efficient production of EPs. According to the result of previous studies and the constructs available in our lab, including pCBD2 (entire DV2 E protein), pCBD2J396 (containing the stem-anchor region of JEV sequences), and pCBD2J442 (containing the anchor region of JEV sequences), we construct pCBD2J396D442 (containing the stem region of JEV sequences) and pCBD2J430 (containing an extended anchor region of JEV in the first part of our study). In the second part of our study, we use site-directed mutagenesis to generate mutant pCBD2 constructs that contain different amino acid substitutions in the stem region. The ability to generate EPs by these prM/E constructs and mutant pCBD2 constructs were examined. The results revealed that neither prM/E constructs containing a single stem or anchor region of JEV nor mutant pCBD2 constructs can generate EPs efficiently.
These findings suggest both the stem and anchor region are important for efficient EPs production. The interaction between the stem and anchor region remains to be investigated in the future.
Efficient production of EPs by prM/E construct of flaviviruses has potential to be employed in future vaccine development and in serodiagnosis. Our study revealed that both the stem and anchor regions are important for efficient EPs production. Although no specific motif or amino acid in the stem-anchor region was identified as critical element for EPs production, investigation of interaction between the stem and anchor regions in the future may provide important information for developing strategies to block the function of this region.
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