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研究生:李宏仁
研究生(外文):Hung-Jen Lee
論文名稱:選殖日本腦炎病毒RNA聚合酶及其抗體製備
論文名稱(外文):Expression of RNA-dependent RNA Polymerase of Japanese Encephalitis Virus and Preparation of Polyclonal Antibody
指導教授:張瑞宜張瑞宜引用關係
指導教授(外文):Ruey-Yi Chang
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:53
中文關鍵詞:日本腦炎
外文關鍵詞:RNA-dependent RNA polymeraseNS5
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黃質病毒為正股單股的RNA病毒,而病毒產生的RNA-dependent RNA polymerase(RdRp)對於黃質病毒RNA基因體複製來說是關鍵的酵素。為瞭解黃質病毒RNA的複製機制,本實驗將日本腦炎病毒非結構蛋白基因NS5經RT-PCR後選殖至表現載體pET-28a(+),並使NS5蛋白N端與histidine-tag形成融合蛋白。當此表現載體轉殖至大腸桿菌BL21 Star(DE3)並以1 mM IPTG誘導可產生大量的重組蛋白。此重組RdRp蛋白可溶於含有0.25% Tween 20的溶液,並以nickel affinity column純化。純化的重組NS5蛋白經注射兔子後得到多株抗體,以西方墨漬法可證明此多株抗體對NS5具有抗原專一性;此外,利用此抗體可進行免疫螢光分析法,可以偵測受日本腦炎病毒感染的細胞。重組RdRp蛋白無法以含有3’端非轉譯區的1.7 kb片段為模板進行RNA複製。其原因可能是重組蛋白質在大腸桿菌系統表現時產生錯誤折疊,或是缺乏轉譯後修飾,而造成重組RdRp蛋白失去酵素活性。
The virus-encoded RNA-dependent RNA polymerase (RdRP), which is required for replication of the positive-strand RNA genome, is a key enzyme of the members of the virus family Flaviviridae. To study the mechanism of flaviviral RNA replication, the RdRP encoded by nonstructural gene NS5 of Japanese encephalitis virus, member of flavivirus, was amplified by RT-PCR and subsequently cloned into an expression vector, pET-28a(+), as a fusion protein with histidine-tag at the N-terminus. High level of recombinant proteins could be detected with induction of 1 mM IPTG when the expression plasmid was transformed into E. coli BL21 Star(DE3). The recombinant RdRp could be solubilized with 0.25% Tween 20 and purified through nickel affinity column. The eluted recombinant NS5 protein was used to immunize rabbits and polyclonal antibody was raised. The antigenicity of polyclonal antibody specific to NS5 was determined by western blotting and by immunofluorescence assay on JEV-infected cells. The enzymatic activity of the recombinant RdRP was sought using 1.7 kb in vitro RNA transcripts containing 3’ untranslated region (UTR) as templates. However, no synthesized complementary strand of these molecules could be detected suggesting that recombinant NS5 lack of enzymatic activity probably due to misfolding or improper post translational modifications when expressed in E. coli system.
一、 中文摘要……………………………………………………… 2
二、 英文摘要……………………………………………………… 3
三、 研究背景及重要性…………………………………………… 4
四、 材料與方法…………………………………………………… 11
五、 結果…………………………………………………………… 25
六、 討論…………………………………………………………… 30
七、 參考文獻……………………………………………………… 35
八、 圖表…………………………………………………………… 40
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