- 學位論文
比較榕樹透翅毒蛾核多角體病毒與黑角舞蛾核多角病毒之 fp25k 基因和其在昆蟲細胞內的表現
Comparison of two fp25k genes from Perina nuda and Lymantria xylina multiple nucleopolyhedroviruses and their expression in insect cells
摘要
核多角體病毒感染細胞株之末期，會產生許多由多角體蛋白所包埋的封埋體病毒，根據細胞內形成多角體的多寡可分為繁多角體 (multiple polyhedra, MP) 和寡多角體 (few polyhedra, FP) 兩種。寡多角體的形成與是否能夠轉譯出 25 kDa 的 fp25k 基因有關。當 fp25k 基因發生缺失或是插入外來的 DNA 片段時，會使病毒在感染細胞後呈現 FP 的現象。榕樹透翅毒蛾核多角體病毒 (Perina nuda multiple nucleopolyhedrovirus, PenuMNPV) 與黑角舞蛾核多角體病毒 (Lymantria xylina multiple nucleopolyhedrovirus, LyxyMNPV) 的 fp25k 基因分別含有 624 與 651 個鹼基對，且榕樹透翅毒蛾核多角體病毒與黃杉毒蛾核多角體病毒 (Orgyia pseudotsugata multiple nucleopolyhedrovirus, OpMNPV) 較為相似；黑角舞蛾核多角體病毒則與吉普賽舞蛾核多角體病毒 (Lymantria dispar multiple nucleopolyhedrovirus, LdMNPV) 較為相似。榕樹透翅毒蛾與黑角舞蛾核多角體病毒的 fp25k 基因皆具有一個晚期和非常晚期基因的轉錄起始點 ATAAG，為桿狀病毒感染過程中晚期表現基因。FP25K 蛋白質經電腦預測發現具有 coiled-coil 和 putative actin-binding domains。根據 fp25k 基因所建構之親緣關係樹發現榕樹透翅毒蛾核多角體病毒屬於 group Ⅰ，而黑角舞蛾核多角體病毒屬於 group Ⅱ 的病毒群。在西方墨點法偵測榕樹透翅毒蛾核多角體病毒的 FP25K 蛋白發現，其為一個結構蛋白並且表現在感染的晚期。以間接免疫螢光法發現榕樹透翅毒蛾核多角體病毒的 FP25K 表現在細胞質與細胞核內。野生型的榕樹透翅毒蛾與黑角舞蛾核多角體病毒皆為 MP 的病毒株。但 LyG (LdMNPV-like virus) 病毒株感染吉普賽舞蛾細胞株後皆呈現 FP 的現象，此病毒的 fp25k 基因序列發生缺失現象，經由電腦分析轉譯結果顯示造成框架移動突變 (frameshift mutation)，在 Ly5 (LyxyMNPV) 病毒株為 MP 與 FP 共存的現象，分析發現具有野生型與缺失的 fp25k 基因，因此 fp25k 基因的突變將會直接影響到桿狀病毒的 FP 現象。
並列摘要
The nucleus of an infected cell by nucleopolyhedrovirus is filled with occlusion bodies (OBs) embeded by polyhedrin in the late infection phase. According to the numbers of polyhedra per cell, two types of cytopathic effect (CPE) can be divided, multiple polyhedra (MP; >10 OBs/cell) and few polyhedra (FP; < 10 OBs/cell). FP phenotype is associated with fp25k gene which encodes 25 kDa. When the fp25k sequence is deleted or inserted by the foreign DNA, it causes reducing numbers of OBs in the infected cells. The fp25k of Perina nuda multiple nucleopolyhedrovirus (PenuMNPV) and Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) contained 624 and 651 base pairs, respectively. The former is closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and the later is to Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Both PenuMNPV and LyxyMNPV fp25k genes are late genes containing a transcriptional initiation site, TAAG, of late and very late gene motifs. FP25K protein possessed with coiled-coil and putative actin-binding domains by computer prediction. A phylogenetic tree was constructed based on fp25k sequences, PenuMNPV and LyxyMNPV belonged to group Ⅰ and group Ⅱ, respectively. FP25K protein was confirmed that it is a structure protein and expressed in late phase by Western blotting hybridization. Both PenuMNPV (Ө52) and LyxyMNPV (Ly2) in our lab strains displayed MP phenotype. LyG (LdMNPV-like virus) from infected Lymantria xylina exhibited FP phenotype in infected cells, and its fp25k was deleted 44 nucleotides comparing with the fp25k of LdMNPV. Ly5 strain exhibited two phenotypes, MP and FP in infected LD cells, its fp25k sequence contained wild and deleted type. Therefore, the expression of fp25k or deleted fp25k directly affected the CPE (MP or FP) phenotype of infected cells.