日本腦炎病毒(Japanese Encephalitis Virus, JEV)是以蚊子為媒介傳播的黃質病毒科成員之一，引起高致死率的日本腦炎(Japanese Encephalitis)。JEV基因體約11kb正性單股RNA，含第一型帽蓋(cap)和5'端和3'端非轉譯區中間為一個大開放性讀框架(ORF)，能轉譯出一段多蛋白，經轉譯後修飾作用產生三個結構性蛋白及七個非結構性蛋白。病毒感染性質體(infectious clone)及複製子(replicon)之建立，可提供研究病毒複製機制及致病原因最強而有力的工具。儘管如此，JEV感染性質體可能因病毒基因或蛋白會對細菌宿主造成毒害，導致病毒基因常會發生重組、刪除等突變的問題，使JEV感染性質體不易建立。本論文研究目的為構築新穎性的JEV感染性質體和複製子，研究策略為分別利用pcDNA3.1/HisC和pBR322為載體、大腸桿菌DH5-α菌株和BD1528菌株為宿主，構築出5’端帶有CMV promoter調控及3’端接上HDV ribozyme的JEV感染性質體與複製子。首先將全長JEV病毒序列分三大段(F1、F2、F3)，依序插入pcDNA3.1/HisC，再轉入大腸桿菌DH5-α菌株選殖。到目前為止已成功將JE-F2F3 (3400nt-10970nt)序列選殖到此載體。然而，JE-F1(1-3600nt)序列發生574-1936nt 刪除突變。以pcDNA3.1/HisC表達載體構築出prM和E刪除突變的JEV序列以提供後續改造感染性質體與複製子的模板。以pBR322構築感染性質體方面，首先要插入含有限制酶(KpnI、NotI、XhoI)切位的linker來改造pBR322質體，將SV40(Simian virus 40) polyA序列接入此質體，構築出pBR322-SV40pA。並以成功將含有67 bp HDV ribozyme序列的JE-F2F3接至pBR322-SV40pA載體。JE-F1(1-3600 nt)片段選殖送入到pBR322-SV40pA，但在741nt有無意義突變(nonsense mutation) 並產生一個終止密碼子。最後以pBR322構築複製子的方面，在pBR322- JE-F1(1-3600 nt)-SV40pA載體上去除大部分的結構性基因去構築JEV複製子，保留C蛋白N端8aa和E蛋白C端30aa，在它們之間插入含FMDV2A的紅色螢光蛋白的基因，複製子之5'-UTR前加入一段CMV promoter及3'-UTR之後接上HDVr序列，期待能建構出穩定表達的JEV複製子。
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.
