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研究生:楊仕音
研究生(外文):Shih-Yin Yang
論文名稱:一種新的100kDa蝦白點症病毒蛋白質之研究
論文名稱(外文):Analysis of a novel 100 kDa protein of shrimp WSSV
指導教授:郭光雄羅竹芳羅竹芳引用關係
指導教授(外文):Guang-Hsiung KouChu-Fang Lo
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:85
中文關鍵詞:白點症病毒反轉錄聚合酵素鏈反應早期表現基因蛋白質表現離體轉錄/轉譯實驗西方轉印雜合實驗穿越細胞膜區域
外文關鍵詞:white spot syndrome virusWSSVRT-PCRearly geneprotein expressionin vitro transcription/translationWestern blot hybridizationtransmembrane domains
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本實驗室建立之白點症病毒 (white spot syndrome virus, WSSV) Hind III 基因庫(library)中,包含一段全長約4.4 kb的基因,命名為pmh15,此片段接入pUC19載體中,已由病毒基因體完成確認與定序工作。Pmh15包含有六個開放譯讀區(open reading frames, ORFs),其中最長的ORF1由2535個去氧核糖核酸所組成,可轉譯成845個氨基酸,蛋白質分子量大小經電腦預測之理論值為92 kDa。推測轉譯起始位置,即methionine start codon的鄰近序列為AGTATGG,符合真核生物轉譯起始原則 (Kozak rule),轉譯終點位置3’方向46核酸處則存在一個polyadenylation consensus signal (polyA signal) AATAAA。經反轉錄聚合酵素鏈反應(reverse transcriptase polymerase chain reaction)進行基因轉錄時序分析,可知ORF1屬於病毒感染之早期表現基因(early gene)。
將包含前258個氨基酸部份之DNA片段以聚合酵素鏈反應 (polymerase chain reaction, PCR) 放大後,接入pQE載體進行活體蛋白質表現(in vivo expression),於大腸桿菌 (E. coli) 中可誘發出一40 kDa的蛋白質產物,並經N端定序確認其氨基酸序列。誘發出的蛋白質純化後注射至紐西蘭白兔體內生產抗體以進行進一步的分析。
Pmh15-ORF1全長則接入pcDNA3 載體,經由離體轉錄/轉譯實驗 (in vitro transcription/translation) 生產出100 kDa之蛋白質。利用生產自白點症病毒100 kDa蛋白質N端之抗體進行西方轉印雜合實驗 (Western blot hybridization) 可偵測到離體轉錄/轉譯之蛋白質產物,以及此100 kDa蛋白質於白點症病毒感染之病蝦組織中確實存在。
而在電腦比對方面,WSSV 100 kDa蛋白質發現有三個推測性的穿越細胞膜區域 (transmembrane domains)及8個可能被醣化(N-glycosylation)與31個可能被磷酸化 (phosphorylation)之位置。然而,WSSV 100 kDa蛋白質並沒有發現與任何桿狀病毒(baculovirus)或其它大型DNA病毒已發表的膜蛋白(envelope protein)及穿越膜蛋白(transmembrane proteins)有任何相似之處,顯示白點症病毒在分類上極有可能為桿狀病毒中新的一屬,或甚至所有病毒種類中新的一科。

WSSV genomic library of Hind III was constructed in pUC19, and contained a 4.4 kb genomic fragment named pmh15. The pmh15 nucleotide sequences have been identified and sequenced from the viral genome. The pmh15 was composed of six open reading frames (ORFs), including the longest ORF─pmh15 ORF1 with 2535 nucleotides. And the pmh15-ORF1 encodes 845 amino acid polypeptide of a theoretical size of 92 kDa from computer predictions. The sequence surrounding the methionine start codon (AGTATGG) conformed with the Kozak rule for efficient eukaryotic translation initiation and a polyadenylation consensus (polyA) signal is present 46 nt downstream of the translational stop codon. RT-PCR was used to investigate the temporal expression of the pmh15-ORF1, the results show that the pmh15-ORF1 gene may express early in the course of WSSV infection.
A DNA fragment containing the partial pmh15-ORF1 was amplified by PCR reaction for cloning from the start codon to amino acid 258, then ligated into pQE for in vivo expression. The partial pmh15-ORF1 was expressed in E. coli, which produced a 40 kDa protein product. And the N terminal amino acids of the expressed protein were sequenced as prediction. The purified protein was injected into a New Zealand white rabbit to produce antibodies for subsequent analysis.
The full-length pmh15-ORF1 was constructed in vector pcDNA3 for in vitro transcription/translation (TNT), and a major product was 100 kDa. The antibody against the N terminus of the WSSV 100 kDa protein could recognize the TNT product and a 100 kDa protein in WSSV-infected shrimp tissues.
The 100 kDa protein contains three possible transmembrane domains, 8 putative N-glycosylation sites and 31 possible phosphorylation sites. This viral protein showed no significant homology to any published envelope or transmembrane proteins of baculovirus and other large DNA viruses, that suggests WSSV could be a representative of a new baculovirus genus (whispovirus) or even a new virus family (Whispoviridae).
中文摘要 1
英文摘要 3
序言 5
材料與方法 10
ㄧ、DNA定序與電腦比對分析 10
二、病毒基因體DNA之萃取 10
三、聚合酵素鏈反應 (PCR) 檢測 11
四、點墨雜合法 (dot hybridization) 12
4.1探針 (probe) 之製備 12
4.2點墨雜合法 13
4.3 Rebrobing 13
五、實驗蝦體之收集、 total RNA的萃取
與實驗蝦體之感染測試 14
5.1 實驗蝦體之收集及total RNA的萃取 14
5.2實驗蝦體之感染檢測 15
5.2.1 DNA之萃取 15
5.2.2白點症病毒聚合酵素鏈反應第一次擴增反應 16
5.2.3白點症病毒聚合酵素鏈反應第二次擴增反應 16
5.2.4 DNA 品質之監控 17
六、反轉錄聚合酵素鏈反應 (RT-PCR) 17
七、pGEM-T easy-pmh15 ORF1的構築 18
7.1 PCR及接合作用(ligation) 18
7.2大腸桿菌勝任細胞之製備 19
7.3大腸桿菌轉型作用(Transformation) 19
八、離體轉錄/轉譯實驗( In vitro transcription/translation ) 20
九、pQE-pmh15 ORF1 N端的構築與蛋白質之誘發與純化 21
9.1 pQE-pmh15 ORF1 N端的構築 21
9.2細菌的誘發(induction of bacteria) 21
9.3蛋白質的純化 (Protein purification) 22
9.4 純化後蛋白質濃度之測定 23
十、抗體之生產 23
十一、西方轉印雜合實驗 (Western blot hybridization) 24
11.1抗體的準備 24
11.2 WSSV感染病蝦組織之置備 24
11.3 西式點漬法 (Western blotting) 24
結果 26
一、 Pmh15 開放譯讀區基因的位置及序列分析 26
二、Pmh15-ORF1 DNA的來源及引子對檢測可靠性的分析 27
三、Pmh15-ORF1之轉錄時序分析及實驗蝦體之感染檢測 28
四、離體轉錄/轉譯 29
五、蛋白質表現及分析 30
討論 32
一、Pmh15-ORF1之基因表現時序分析 32
二、蛋白質表現及分析 32
三、氨基酸定序 35
四、Western式點漬法 35
五、WSSV 100 kDa蛋白質氨基酸序列
與其他蛋白質之相似度比對 36
結論 39
參考文獻 40
圖表
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