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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王重雄(Chung-Hsiung Wang) | |
| dc.contributor.author | Yu-Shin Nai | en |
| dc.contributor.author | 乃育昕 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:24:47Z | - |
| dc.date.available | 2008-07-28 | |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-22 | |
| dc.identifier.citation | Ahmad, M., S. M. Srinivasula, L.Wong, G. Litwack, T. F. Alnemri, and E. S.
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K.. 1997b. Regulation of programmed cell death by baculovirus. pp. 237-266. In: The Baculoviruses. Plenum Press, New York and London. Monique, M. V. O., H. C. A. Marleen, A. H. Elisabeth, C. W. G. Joost, P. Sander, and M. V. Just. 2005. Genome sequence of Chrysodeixis chalcites nucleopolyhedrovirus, a baculovirus with two DNA photolyase genes. J. Gen. Virology. 86: 2069-2080. Pei, Z., G. Reske, Q. Huang, B. D. Hammock, Y. Qi, and N. Chejanovsk. 2002. Characterization of apoptosis suppressor protein P49 from the Spodoptera littoralis nucleopolyhedrovirus. J. Biol. Chem. 277: 48677-48684. Seshagiri, S., and L. K. Miller. 1997. Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1. Cell Biol. 94: 13606-13611. Thiem, S. M., and N. Chejanovsky. 2004. The role of baculovirus apoptotic suppressors in AcMNPV-mediated translation arrest in Ld652Y cells. Virology. 319: 292-305. Verhagen, A. M., E. J. Coulson, and D. L. Vaux. 2001. Inhibitor of apoptosis protein and their relatives: IAPs and other BIRPs. Genome Biology 2 (7): 3009.1-3009.10. Wilfred, F. J., I. J. Kel, A. V. S. Elisabeth, G. M. H. Jaconus, B. Rene, Z. Douwe, W. G. Rob, and M. V. Just. 1999. Sequencr and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. J. Gen. Virology. 80: 3289-3304. Wu, C. Y., and C. H. Wang. 2005. Characterization and polyhedrin gene cloning of Lymantria xylina multiple nucleopolyhedrovirus. J. Invertebr. Pathol. 88: 238-246. Yin, X. M., and Z. Dong. 2003. Analysis of Apoptosis: basic principles and procedures. pp. 239-251. In: Essentials of apoptosis. Humana Press. New Jersey. Zanotto, P. M., B. D. Kessing, and J. E. Maruniak. 1993. Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. J. Invertebr. Pathol. 62: 147-164. Zarudnaya, M. I., I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun. 2003. Downstream elements of mammalian pre-mRNA polyadenylation signals: primary, secondary and higher-order structures. Nucleic acids Res. 31: 1375- 1386. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33094 | - |
| dc.description.abstract | 昆蟲桿狀病毒感染非寄主昆蟲細胞，即可以觀察到細胞凋亡的現象。昆蟲桿狀病毒為能夠順利在感染昆蟲細胞達到增殖的目的，其基因組內主要含有兩種抑制細胞凋亡之基因，即為p35 和iap 基因群(inhibitor of apoptosis gene family, iap1~iap5)，其中iap 基因較p35 基因廣泛地分布於桿狀病毒中。經PCR 增幅再利用南氏墨點法證實，黑角舞蛾核多角體病毒(Lymantria xylina multiple nucleopolyhedrovirus, LyxyMNPV) 之基因組內含有兩個iap 基因，分別為ly-iap2 (687 bp) 與ly-iap3 (450 bp)，而不具有加州苜蓿夜蛾核多角體病毒(Autographa californica MNPV, AcMNPV) 之iap1 及p35 同源基因。此外黑角舞蛾核多角體病毒之iap 基因與吉普賽舞蛾核多角體病毒(L.dispar MNPV) 之iap 基因皆呈高相似度，iap2 及iap3 分別為81% 及86%。利用北方雜合法證實黑角舞蛾核多角體病毒ly-iap2 在細胞感染72 小時後，即可偵測到一1.95-kb 的轉錄區；而ly-iap3 在感染48 小時後，可偵測到兩個轉錄區，分別為1.04-kb 及2.09-kb。黑角舞蛾核多角體病毒的兩個iap 基因和榕樹透翅毒蛾核多角體病毒(Perina nuda MNPV, PenuMNPV) 的相同iap 基因均已解序，據此序列與其他桿狀病毒之相同基因序列分析親緣關係，結
果顯示LyxyMNPV 之iap 基因與LdMNPV 關係密切，而與PenuMNPV 和 AcMNPV 皆處於不同群中。LY-IAP3 經表現後，並成功製造出抗血清，利 用間接螢光免疫偵測法(indirect fluorescen immunoassay, IFIA)，LY-IAP3 蛋白質在LyxyMNPV 感染之不接受細胞株(NTU-SL7B) 以及可接受細胞株(IPLB-LD 652Y-7) 後之表現，發現LY-IAP3 皆可在SL7B 或LD7 細胞中表現。因此LY-IAP3 在病毒感染過程中所扮演的角色，需更進一步釐清。 | zh_TW |
| dc.description.abstract | The anti-apoptosis genes of baculoviruses are essential genes for viral propagation in their susceptible host cells. There are two main groups of antiapoptotic genes in baculovirus genome, p35 and iap gene family (iap1~iap5), the latter is more wildly distributed in baculovirus. According to the results of PCR amplification and Southern blot hybridization, Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) contains only two iap genes ly-iap2 (687 bp) and ly-iap3 (450 bp). The sequences of iap2 and iap3 of LyxyMNPV had high identities with those of L. dispar MNPV (LdMNPV), about 81% and 86%, respectively. The results of Northern blot hybridization revealed that, a 1.95-kb transcript of ly-iap2 was first detected at 72 h postinfection (pi) and 1.04- and 2.09-kb transcripts of ly-iap3 were first detected at 48 h pi. The iap genes of Perina nuda MNPV (PenuMNPV) had been also cloned and sequenced. LyxyMNPV and PenuMNPV were compared with other known baculovirus iap genes for phylogenetic analysis. The trees showed that LyxyMNPV was closely related to LdMNPV, in addition, LyxyMNPV, PenuMNPV and Autographa californica MNPV (AcMNPV) were located seperately in three different clusters. LY-IAP3 polyclonal antibody had been prepared and used to detect the expression of LY-IAP3 in LyxyMNPV-infected IPLB-LD 652Y-7 cells (LyxyMNPV permissive cells) and NTU-SL7B cells (LyxyMNPV non-permissive cells) by indirdct fluorescence immunoassay (IFIA). LY-IAP3 could be expressed in the cytoplasm of both cells; therefore, the role of LY-IAP3 needs to be further elucidated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:24:47Z (GMT). No. of bitstreams: 1 ntu-95-R93632010-1.pdf: 1191704 bytes, checksum: 67be4191aca48369debc96ea16afe399 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | i
中文摘要……………………………………………………………………………....................… 1 英文摘要……………………………………………………………………....................………… 2 壹、緒言………………………………………………………………………....................……… 3 貳、往昔研究……………………………………………………………...................…………… 7 參、材料與方法………………………………………………………….………...............…… 10 一、供試昆蟲………………………….........………………………..........………………… 10 二、供試細胞株……………………………………………………..................…………… 10 三、供試病毒株……………………………………………………..................…………… 10 四、病毒感染………………………………………………………………...................…… 10 五、包埋體與病毒粒子之純化…………………………………………..............……… 11 六、病毒DNA 之製備……………………………………………………...................…… 11 七、限制?之切割…………………………………………………………...................…… 12 八、PCR-RFLP…………………………………………………………..................……… 12 九、增幅榕樹透翅毒蛾核多角體病毒之iap 基因……………….........…………… 12 十、探針製備………………………………………………………..................…………… 13 十一、南氏墨點法……………………………………………….................……………… 13 1. 轉印…………………………………………………………….....................…………… 13 2. 雜和反應………………………………………………………....................…………… 14 3. 呈色反應…………………………………………………………....................………… 14 十二、基因選殖………………………………………………….....................…………… 15 十三、轉型作用……………………………………………………………..................…… 15 十四、質體DNA 之萃取……………………………………………..................………… 15 十五、RNA 之製備……………………………………………………...................……… 16 十六、cDNA 之製備………………………………………………….................………… 16 十七、3’RACE……………………………………….................………………………… 17 十八、北方雜合法…………………………………..................…………………………… 17 1. 轉印………………………………………………….……….....................……………… 17 ii 2. 雜和反應……………………………………………………….....................…………… 17 3. 呈色反應……………………………………………………….....................…………… 18 十九、基因表現……………………………………………………………..................…… 18 二十、聚丙烯醯胺膠體電泳( SDS-PAGE ) ………………………………..........… 19 二十一、蛋白質的時序性表現(Time course) …………………………....……… 20 二十二、西方墨點雜合法(Western blotting hybridization) …………… 20 二十三、間接免疫螢光偵測法…………………..............……………………………… 20 肆、結果………………………………………………….................…….……………………… 21 一、PCR-RFLP……………………………………………………..................…………… 21 二、細胞凋亡形態觀察………………………………………………………................… 21 三、黑角舞蛾核多角體病毒（Ly2）抗細胞凋亡基因之選殖………...………… 21 四、ly-iap3 基因之3’ 端加聚腺嘌呤(poly A) 位置之分析………………….22 五、蛋白質結構分析…………………………………................……………………….… 22 六、榕樹透翅毒蛾核多角體病毒iap 基因之選殖………………......…………….…23 七、LyxyMNPV、MaviMNPV 及PenuNPV 與其他桿狀病毒iap 基因和蛋白 質相似度比較…………………………………………………….........…….........…… 23 八、親緣關係之分析（phylogenetic analysis) …………………………………24 九、北方墨點法………………………………………………………………..................… 25 十、重組蛋白質表現與純化………………………………………………...............…… 25 十一、間接免疫螢光偵測法………………………………………...............…………… 25 伍、討論……………………………………………………………………..................………… 27 陸、參考文獻………………………………………………………………................………… 32 柒、圖表…………………………………………………………………….................………… 37 致謝…………………………………………………………………………..................………… 69 附錄一、建構演化樹所使用各種核多角病毒之GenBanK 的accession number…................................................................................................... 70 附錄二、本實驗中所使用的引子名稱與序列………………………………….......…… 72 iii 表次 表一核多角體病毒iap1 基因序列之相似度……………………………….........……… 37 表二核多角體病毒IAP1 蛋白質序列之相同度與相似度………….....……………… 38 表三核多角體病毒iap2 基因序列之相似度………………………………….........…… 39 表四核多角體病毒IAP2 蛋白質序列之相同度與相似度………………….....……… 40 表五核多角體病毒iap3 基因序列之相似度…………………………………........……. 41 表六核多角體病毒IAP13 蛋白質序列之相同度與相似度…………………………...42 iv 圖次 圖一聚合?鏈鎖反應擴增多角體蛋白基因片段與限制?切割反應………….......… 43 圖二黑角舞蛾病毒株感染不同細胞株產生的細胞病變……………………….......… 44 圖三增幅ac-p35、ac-iap1、ly-iap2 及ly-iap3 部分或全長基因片段…. 45 圖四南氏墨點法偵測黑角舞蛾核多角體病毒iap2 基因之限制?圖譜………...… 47 圖五南氏墨點法偵測黑角舞蛾核多角體病毒iap3 基因之限制?圖譜………...… 48 圖六黑角舞蛾核多角體病毒iap3 基因與胺基酸序列……………………….....….… 49 圖七黑角舞蛾核多角體病毒IAP 胺基酸序列結構…………………………….........… 51 圖八增幅榕樹透翅毒蛾核多角體病毒之iap1、iap2 及iap3 基因……………… 53 圖九核多角體病毒iap1 序列之親緣關係樹…………………………………….........… 54 圖十核多角體病毒iap2 序列之親緣關係樹………………………………….........…… 56 圖十一核多角體病毒iap3 序列之親緣關係樹………………………………........…… 58 圖十二北方墨點法分析感染黑角舞蛾核多角體病毒之細胞中iap2 基因之表現60 圖十三北方墨點法分析感染黑角舞蛾核多角體病毒之細胞中iap3 基因之表現61 圖十四利用原核表現系統表現黑角舞蛾核多角體病毒之IAP3 重組蛋白……… 62 圖十五聚丙烯醯胺膠體電泳和西方墨點法偵測純化後之IAP3 重組蛋白……… 63 圖十六間接免疫螢光偵測黑角舞蛾核多角體病毒IAP3 在細胞中的表現位置…64 圖十七間接免疫螢光偵測黑角舞蛾核多角體病毒IAP3 在LD7 細胞中之表現時 序……..................………………………………………………………………………… 65 圖十八間接免疫螢光偵測黑角舞蛾核多角體病毒IAP3 在SL7B 細胞中之表現時 序…..……………………………………………………………..................................…67 | |
| dc.language.iso | zh-TW | |
| dc.subject | 黑角舞蛾核多角體病毒 | zh_TW |
| dc.subject | 抑制細胞凋亡基因 | zh_TW |
| dc.subject | iap | en |
| dc.subject | LyxyMNPV | en |
| dc.title | 黑角舞蛾核多角體病毒抑制細胞凋亡基因iap2 及iap3 之選殖、定序、表現及比較 | zh_TW |
| dc.title | The study of anti-apoptotic genes iap2 and iap3 of Lymantria
xylina multiple nucleopolyhedrovirus, (LyxyMNPV): cloning, sequencing, expression and comparison | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張俊哲,羅竹芳,侯豐男,路光暉 | |
| dc.subject.keyword | 黑角舞蛾核多角體病毒,抑制細胞凋亡基因, | zh_TW |
| dc.subject.keyword | LyxyMNPV,iap, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-22 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
| 顯示於系所單位： | 昆蟲學系 | |
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