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研究生:李俊志
研究生(外文):Chun-Chih Li
論文名稱:嚴重急性呼吸道症候群之病患抗SARS冠狀病毒小外套蛋白抗體反應與病毒結構蛋白間交互作用之研究
論文名稱(外文):A Study of the Antibody Reactivity to SARS-CoV Small Envelope Protein in SARS patients and Interaction Between SARS-CoV Different Structural Proteins
指導教授:陳宜民陳宜民引用關係
指導教授(外文):Yi-Ming A.Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:公共衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:嚴重急性呼吸道症候群
外文關鍵詞:SARSE protein
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嚴重急性呼吸道症候群(SARS)是一種新興的傳染性疾病,其致病源是由一種新型的冠狀病毒(SARS-CoV)所引起。SARS冠狀病毒具有四種結構蛋白,包括:刺釘蛋白(S) 、膜醣蛋白(M) 、小外套蛋白(E)及核外殼蛋白(N)。其中E蛋白(Small envelope protein)為一含有76個胺基酸之膜蛋白,其在病毒的組裝及複製中可能扮演重要的角色。因此,本研究希望了解SARS病患中,產生抗E蛋白-抗體的情形及E蛋白與N蛋白之間的交互作用。
首先,我們利用PCR的方法選殖出SARS冠狀病毒的E蛋白基因,構築了兩個質體─pGEX4T-1-E及 pET29a-E,並以大腸菌表現GST-E及His-E融合蛋白。接著,利用以GST-E蛋白為抗原之西方墨點法,篩檢出38位SARS病人的血清,結果發現其中有1位(No.#27)具有抗E蛋白-抗體反應;再以此病患不同時間點之血清作西方墨點法,發現其抗E蛋白-抗體在發病後第25天開始出現。爲了要印證此病患確實具有抗E蛋白-抗體,我們利用GST-E融合蛋白去製備抗E蛋白-抗兔血清,藉此建立抗體競爭性酵素聯結免疫分析法。結果發現不論是與正常人的血清樣本、或是與其他SARS陽性病患(西方墨點法檢測E為陰性之病患)比較,此位病患的血清樣本確實具有與兔抗血清競爭的效應。類似結果也在競爭性的西方墨點法得到印證。因此判斷此位病患確實具有抗E蛋白-抗體。
我們根據過去對於冠狀病毒的研究,首先推測E蛋白對於N蛋白在病毒蛋白的運送或組裝上可能扮演重要角色,為了證實此一假設,本研究以pGEX4T-1-N、pET28a-N表現質體以大腸菌表現出GST-N與His-N融合蛋白,並以此建立pull down assay。實驗結果顯示,不論以GST-N與His-E的GST-pull down assay,或是以His-N與GST-E的Ni-resin pull down assay,皆未發現兩蛋白間具有交互作用,因此推論E蛋白對於N蛋白並無直接的影響。
Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV). SARS-CoV contains four structural proteins- spike(S), membrane(M), small envelope(E), and nucleocapsid(N)proteins. SARS-CoV E protein is a membrane protein with a size of 76 amino-acids. The goals of this study were 1. To study the anti-E antibody reactivity in SARS patients; and 2. To analyze the interaction between SARS-CoV E and N proteins.
Two plasmids- pGEX4T-1-E and pET29a-E were constructed to express recombinant fusion proteins (GST-E and His-E) in Escherichia coli (E. coli). Western blot assay (WB) with recombinant GST-E protein was used to analyze anti-E antibody reactivity among 38 patients with SARS. The results showed only one patient-No.#27 had anti-E antibody reactivity. Multiple time points of serum samples from patient No. 27 were further analyzed by WB and the results showed that the patients did not have anti-E antibody until 25 days after the disease onset. Rabbit anti-GST-E antiserum was generated and used for a competitive enzyme immunoassay (CEIA). In comparison with 3 normal controls and other SARS patients without anti-E antibody activity, the serum sample from patient No. #27 had competitive effect with the rabbit anti-E antibody. Similar results were obtained in a competitive WB using recombinant GST-E protein.
To elucidate whether SARS-CoV E will interact with N proteins, two fusion protein GST-N and His-N were produce in E. coli and purified for pull-down assay. The result showed that there was no interaction between E and N recombinant proteins using either glutathione-beads or Ni-resin pull down assay.
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