Expression of the Domain III of Japanese Encephalitis Virus Envelope Protein with BaMV satellite-Based Vector in Plants

• Main Authors: Ming-Juan Chen, 陳明絹
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/19431576715511609755

碩士 === 國立中興大學 === 生物科技學研究所 === 92 === Japanese encephalitis (JE) caused by the Japanese encephalitis virus (JEV) is spread by infected mosquitoes in Asia. It can affect the central nervous system and cause severe complications, even death. JEV, a member of the Flaviviridea family, is a single-stranded, positive-sense RNA virus with a genome size of approximately 11Kb. The JE vaccine currently used is a mouse brain-derived, formalin-inactivated JEV. This JE vaccine has many disadvantages. Thus, the development of JE vaccine that is safer, less costly and effective is important. A promising strategy for developing subunit vaccines involved in the expression of recombinant vaccine immunogens in edible plants, which can be grown and produced easily. There are many advantages in this oral vaccine than the conventional vaccine. Bamboo mosaic virus (BaMV), a member of the potexvirus, contains a single-stranded, positive sense RNA genome with a 5’ cap structure and 3’ poly (A) tail. A satellite RNA (satBaMV) associated with BaMV is a linear RNA molecular of 836 nts [excluding the poly (A) tail] encoding a non-structure protein of 183 amino acids (P20). The BaMV and satBaMV vector systems have been developed to express foreign genes by replacing the ORF of satBaMV. In this study, the coding sequence of domain III of JEV envelope protein cloned into the satBaMV vector can express in N.benthamiana inoculated with the helper virus infectious cDNA clone pCB. Various constructs were designed to compare the efficiency of expression, such as the constructs either fusion with c terminus of P20 or 50 amino acids of P20 c-terminus or not (ie. pCBSJ, pCBSJ-f50aa, and pCBSJ-P20). After inoculation of plants with pCB and each recombinant satellite construct, the replication of chimeric satellite and translation of protein were determined by Northern and Western blot analyses. Besides, the JEV gene was introduced into plants by Agrobacterium transformation. After challenge with BaMV, the transcripts of satellite cassette containing the sequence of the domain III of JEV E protein were amplified. Expression level of the domain III of JEV envelop protein induced by BaMV infection was significantly high than transgene expression.