Construction of infectious clones and replicons ofJapanese encephalitis virus

• Main Authors: Yu-Chun Chang, 張祐鈞
• Other Authors: 林振文
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/40575358887391541603

碩士 === 中國醫藥大學 === 醫學檢驗生物技術學系碩士班 === 100 === Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae that cause Japanese encephalitis with a high fatality rate. JEV genome is an approximately 11kb single-stranded positive sense RNA, containing a type I cap and a single large open reading frame (ORF) flanked by 5’and 3’ untranslated regions (UTRs). This ORF encodes a single polyprotein as three structure proteins and seven nonstructural (NS) proteins by the proteolytic process. Viral infectious clones and replicons are powerful tools for studying the viral replication and pathogenesis. However, constructing JEV infectious clones is difficult due to recombination and deletion events. The study aimed to construct novel JEV infectious clones and replicons, JEV infectious clones and replicons fused with CMV promoter at 5’end and HDV ribozyme at 3’ end were constructed based on high-copy number vector (pcDNA3.1/HisC) and low-copy number vector (pBR322) and transformed in E.coli DH5α and BD1528. Firstly, three fragments(F1、F2、F3) of the full-length JEV genome were cloned into pcDNA3.1/HisC vector, respectively. Up to now, F2 and F3 fragments have been in-frame cloned into pcDNA3.1/HisC. However, the JE-F1(1-3600) occurred a deletion in 574-1936nt. This deleted clone supply as template for further construction of JEV infectious clones and replicons. On the other hands, a modified pBR322 vector with restriction enzyme site linker and SV40 polyA was used for constructing pBR322-SV40pA plasmid. The JEV F2-F3fragment fused with 67bp HDV ribozyme sequence at 3’-end is successfully inserted into this vector and grown in E.coli BD1528. But, JE-F1(1-3600 nt) clone appeared a nonsense mutation in the prM protein. For constructing replicons, most of structure protein gene was deleted, the gene order in replicons was 5’-CMV promoter-JEV5’-UTR-8 amino acids of Capsid protein (nucleotides 96-119)-DsRed2 fluorescent protein-foot and mouth disease virus 2A self-cleaving protease (FMDV2A)- retain the 30 amino acids of Envelope protein (nucleotides 2388-2477), NS1-NS5 proteins- 3’-UTR and 67bp HDVr sequence. We expect that these JEV replicons exhibit a significant biological function.