王重雄臺灣大學：昆蟲學研究所潘秀雯Pien, Hsiu-WenHsiu-WenPien2007-11-262018-06-292007-11-262018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/55052以黑角舞蛾核多角體病毒 (Lymantria xylina multiple nucleopolyhedrovirus; LyxyMNPV) 感染吉普賽舞蛾細胞株 (IPLB-LD652Y-5d) 後，發生細胞融合形成一大的多核細胞，此細胞融合現象可能與病毒套膜融合蛋白有關。核多角體病毒的套膜蛋白質包括 gp64 與 ld130 基因，此兩個蛋白質在低 pH 值的誘導下，會導致感染的細胞融合。第一群核多角體病毒的套膜融合蛋白為 GP64；第二群病毒則為 LD130。黑角舞蛾核多角體病毒之 ld130 基因全長為 2,025 個鹼基對。黑角舞蛾核多角體病毒與吉普賽舞蛾核多角體病毒的 ld130 之核苷酸與胺基酸序列，相似度分別為 93 與 96 %。電腦分析黑角舞蛾核多角體病毒的 LD130 蛋白質序列，發現具有一訊息肽 (signal peptide) 和穿膜區域 (transmembrane domain)，預測其為一膜蛋白。黑角舞蛾核多角體病毒 ld130 基因轉錄本 (transcript) 之分析顯示，其 5’ 端序列具有早期和晚期轉錄起始點。而在 3’ 端的聚腺嘌呤序列位於加聚腺嘌呤訊號 (ATTAAA, poly A signal) 下游第 17 個核苷酸的位置。利用 ld130 基因序列進行親緣關係的分析，黑角舞蛾核多角體病毒屬於第二群病毒。以西方墨漬法偵測 LyxyMNPV 的LD130 蛋白，發現 LD130 為一結構蛋白。以桿狀病毒表現系統 (baculovirus transient expression vector) 極早期基因啟動子所構築之重組 ld130 進行暫時性表現，顯示 LD130 蛋白質的表現和細胞融合有關，並且在酸的誘導下會促使細胞融合。此結果確定了黑角舞蛾核多角體病毒具有 LD130 之套膜融合蛋白，且功能上可能與核多角體病毒第二群之 LD130 相同，為酸誘導後促進細胞融合的重要蛋白質。IPLB LD-652Y-5d cells infected with Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) were found that the infection cells were fused together and formed syncytial giant cells. This phenomenon is probably caused by the viral envelope fusion protein in the cell membrane of the infected cells. Recently, two viral genes encoding the envelope fusion proteins, GP64 and LD130, had been identified. Both GP64 and LD130 envelope fusion proteins mediate the cell fusion at low pH value. The envelope fusion protein of NPV Group I is GP64 while Group II is LD130. We cloned and sequenced ld130 from LyxyMNPV. The LyxyNPV ld130 consists of 2,025 bp. Comparison of the nucleotide and amino acid sequences of LyxyMNPV ld130 with those of LdMNPV ld130 showed 93 and 96% identities, respectively. The predicted peptide sequence of LyxyMNPV LD130 contains signal and transmembrane domains that are significant homology to membrane proteins. Analysis of LyxyNPV ld130 transcript revealed both early and late transcriptional initiation sites located at upstream of the 5’ ATG initiation codon. In addition, a poly A sequence located at 17 nt downstream of the 3’ polyadenylation signal (ATTAAA). Based on the sequences of ld130 gene, the NPV species can be divided into two Groups, I and II, in phylogenetic analysis, LyxyMNPV belongs to Group II. The LD130 of budded virus detected by Western blot revealed that LD130 is a viral structural protein. The LD130 were constructed with the promoter of immediately early gene and expressed by baculovirus transient expression vector in uninfected insect cells showed that LD130 could mediate the cell-to-cell fusion at low pH. These results suggest that a functional homolog of LD130 envelope fusion protein in group II NPVs was found in LyxyMNPV.目錄 中文摘要...............................................1 英文摘要...............................................2 壹、緒言...............................................3 貳、往昔研究...........................................5 參、材料與方法.........................................9 一、供試細胞株.........................................9 二、供試病毒...........................................9 三、核多角體病毒封埋體及病毒粒子之純化.................9 四、出芽病毒之純化................................10 五、病毒 DNA 之純化...............................10 六、PCR-RFLP......................................10 七、南氏墨點法 (Southern blot)........................11 1. 南氏轉印法..................................11 2. 探針 (probe) 之製備.........................11 3. 雜合反應....................................12 八、Total RNA之萃取...................................12 九、快速增殖cDNA末端 (Rapid amplification of cDNA ends, RACE).................................................13 十、親緣關係之分析 (phylogenetic analysis) 及序列相似度之分析....................................................14 十一、大腸桿菌基因表現系統生產重組蛋白................14 1. 質體構築.....................................14 A. 聚合酶鏈反應................................14 B. 勝任細胞 (competent cell) 之製備............15 C. 微量質體DNA萃取法 (plasmid mini-preparation)15 D. 大腸桿菌之轉型作用 (transformation..........16 2. 蛋白質誘導表現與純化.........................16 十二、聚丙烯醯胺膠體電泳 (SDS-PAGE)...................17 十三、西方墨漬法 (Western blot).......................17 十四、不同 pH 對於細胞膜融合的影響....................18 十五、細胞轉染 (transfection).........................18 1. 質體構築.....................................18 2. 轉染作用.....................................18 肆、結果..............................................20 一、PCR-RFLP......................................20 二、感染黑角舞蛾核多角體病毒之細胞病變............20 三、南氏墨點法與基因選殖..........................20 四、5’ 端轉錄起始點和 3’ 端加聚腺嘌呤 (poly A) 位置 之分析........................................20 五、蛋白質結構分析................................20 六、親緣關係之分析 (phylogenetic analysis) 與序列相似 度............................................21 七、重組蛋白質表現與純化..........................21 八、以西方墨漬分析受感染的吉普賽舞蛾細胞之 LD130..21 九、以西方墨漬法偵測出芽病毒......................22 十、pH 值對於細胞膜融合的影響.....................22 十一、細胞轉染 (transfection).....................22 伍、討論..............................................23 陸、參考文獻..........................................28 柒、圖表..............................................34 致謝..................................................48 附錄..................................................49 表次 表一 比較核多角體病毒 ld130 基因序列之相似性.........34 表二 比較核多角體病毒 LD130 蛋白質序列之相同 性及相似性......................................35 表三 不同 pH 值對細胞融合的影響......................36 圖次 圖一 聚合酶鏈反應擴增多角體蛋白基因片段與限制 酶切割反應......................................37 圖二 感染黑角舞蛾核多角體病毒之細胞融合現象..........38 圖三 南氏墨點法偵測黑角舞蛾核多角體病毒 ld130 基因之限制 酶圖譜..........................................39 圖四 黑角舞蛾核多角體病毒 ld130 基因之序列...........40 圖五 黑角舞蛾核多角體病毒 LD130 胺基酸序列結構.......42 圖六 核多角體病毒 ld130 序列之親緣關係樹.............43 圖七 聚丙烯醯胺膠體電泳和西方墨漬法偵測純化後之 LD130 重組蛋白........................................44 圖八 西方墨漬分析感染 LyxyMNPV 之吉普賽舞蛾細胞的 LD130...........................................45 圖九 西方墨漬分析偵測純化的出芽病毒之 LD130..........46 圖十 基因轉染 IPLB-LD652Y-5d 後細胞融合現象..........473280364 bytesapplication/pdfen-US黑角舞蛾核多角體病毒套膜融合蛋白細胞融合Lymantria xylina nucleopolyhedrovirusenvelope fusion proteinld130thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55052/1/ntu-94-R91632004-1.pdf