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研究生:王道元
研究生(外文):Dau-Yuan Wang
論文名稱:竹嵌紋病毒複製酵素蛋白質複合體之研究
論文名稱(外文):Analysis of BaMV RNA Dependent RNA Polymerase Complex
指導教授:徐堯煇
指導教授(外文):Yau-Heiu Hsu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:49
中文關鍵詞:竹嵌紋病毒
外文關鍵詞:BaMV
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竹嵌紋病毒( Bamboo mosaic virus , BaMV )為一正股RNA 病毒,基因體全長約6.4 kb不包含poly(A) tail,包含五個轉譯架構( Open reading frame ),其中ORF1能產生一個病毒核酸複製酵素,包含戴帽酵素( capping enzyme )、類解螺旋酵素( Helicase )及RNA聚合酵素功能區( RdRp ),可複製出病毒基因體。但利用大腸桿菌表達之核酸複製酵素( RdRp domain ),無法和它種病毒一樣具有高轉錄活性,所以推測病毒在感染植物後,會被進一步修飾或是和寄主因子形成蛋白質複合體才具有功能。由於病毒感染細胞後,複製酵素會嵌入特定胞器膜中進行複製,所以本實驗首先對於複製酵素( RdRp )所存在之細胞膜層進行純化,接著以Blue Native PAGE 分析病毒感染與否所形成的蛋白質複合體之差異,發現病毒感染後會形成二個新的蛋白質複合體大小約400 和 600 kDa,經由蛋白質組成之分析,我們認為蛋白質複合體400應該會參與病毒的蛋白質轉譯,且包含putative DEAD-box ATP-dependent RNA helicase 43 、teosinte glume architecture 1。為了確定病毒複製酵素蛋白質複合體之大小,利用膠體過濾層析法所純化之結果發現,RdRp活性所存在的複合體分子量遠高於1500 kDa。接著利用二維電泳去分析膠體過濾層析法所純化之RdRp 複合體,與健康對照組進行分析比對後發現有許多蛋白質增生,可能為病毒複製之寄主因子,參與RdRp 功能。進一步利用辨識戴帽酵素、RNA聚合酵素的抗體進行西方點墨法分析,皆可發現大約50以及150 kDa的訊號,而健康對照組則無;再利用辨識RNA聚合酵素的抗體進行免疫沉澱,沉澱的蛋白質經二維電泳分離後利用西方墨點法分析,也可在pH 6.0以及50 kDa附近偵測到訊號,推測部份BaMV ORF1的三個domain會被切開。最後針對BaMV 外鞘蛋白進行西方墨點法分析,可以觀察到外鞘蛋白在RdRp 活性區有增強之趨勢。總結以上,BaMV 複製酵素蛋白質複合體為一個分子量遠高於1500 kDa,且包含RNA模版之蛋白質複合體,而病毒之核酸複製酵素,可能被切解成至少兩個部份後再和寄主因子、病毒鞘蛋白結合,才能夠在植物內具有功能。而利用二維電泳之分析,我們們也找到了可能參與病毒之複製之寄主因子membrane associated Ca2+ dependent kinase C。
Bamboo mosaic virus (BaMV) has monopartite, positive-strand RNA genome encoding five open reading frames (ORFs). The first ORF encodes the protein that contains capping enzyme, putative RNA helicase domain and RNA dependent RNA polymerase domains for viral genome replication. Unlike few other viral RdRp domains for viral genome replication, BaMV RdRp expressed from E. coli accessed only the low activity for replication, suggesting that the protein may be further processed or need host factors participating in and then fulfills the RdRp function for replication. Previous studies had shown that BaMV replicase same as other viruses of alpha superfamily, is associated with cellular membrane. The membrane containing fraction P30 was first analyzed by Blue Native PAGE, In this gel system, two proein complexes with molecular weights about 400 and 600 kDa induced by viral infection were revealed, we had identified the components of C400, Putative DEAD-box ATP-dependent RNA helicase 43 and teosinte glume architecture 1 might participate in viral protein translation. Further analysis by gel filtration and RdRp assay revealed that the RdRp complex was larger than 1500 kDa. Analysis of the replicase complex purified gel filtration by 2-dimension SDS PAGE revealed several proteins that were induced by viral infection, indicating these host factors may involve in viral replication . Using western blot analysis with antibodies against the methyltransferase and RdRp domains respectively showed that the RdRp fractions contained proteins of 50 and 150 kDa. Using immuno-precipitation and western blot analysis also detected RdRp domain of 50 kDa, Western blot analysis with BaMV coat protein antibody, showed the tendency of increasing the coat protein level among RdRp fractions. In conclusion, the RNA template containing BaMV RdRp complex has molecular weight larger than 1500 kDa, and the ORF1 protein might be processed into at least two parts and then associated with RNA template, coat protein for viral replication. One replicase host protein membrane associated Ca2+ dependent kinase C, might interact with the replicase methyltransferase domain to affect virus replication.
Contents
Chinese Abstract.................................................................................................Ⅰ
English Abstract.................................................................................................Ⅱ
Content................................................................................................................1
Introduction........................................................................................................3
Materials and methods.......................................................................................9
1. Purification of plant membrane protein...................................................9
Purification of P30
RdRp activity assay and analysis of products
2. Protein complexes analysis in P30............................................................10
Blue Native PAGE
Secondary SDS PAGE
Purification and recovery of membrane protein complex
Silver staining
3. Purification of BaMV RdRp subcellular fractions....................................11
Purification of BaMV replication complex by Sephacryl S300
Purification of BaMV replication complexby Sephacryl S1000
4.Western blot analysis of replicase and cp in ubcellular fraction.....13
5. Detection of BaMV RNA ..............................................................................13
Preparation of RNA prob
RNA extraction from BaMV RdRp subcellular fraction Northern blot analysis
6. Two-dimensional SDS PAGE to analyze differential expressed protein...14
Protein precipitation
First dimension isoelectric focusing
Second dimension SDS PAGE
7. Detection of BaMV RNA dependent RNA polymerse...............................15
Immunoprecipitation
Second dimension SDS PAGE
Western blot
Result.....................................................................................................................17
a. Purification of protein complex from membrane protein fraction (P30)
b. Two protein complexes that induced by BaMV infection in P30
c. Further purification of P30 for BaMV replicase subcellular fraction by SEC
d. Two dimensional analysis of differential expressed proteins induced by BaMV infection
e. Identification of replication complex component in BaMV replicase subcellular fractionf. Full length and smaller sized replicase form complex with BaMV coat protein and host kinase
Discussion...............................................................................................................23
Tables and figures..................................................................................................34
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