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研究生:黃建喬
研究生(外文):Chien-Chiao Huang
論文名稱:B型肝炎病毒e抗原結合至嗜中性白血球之序列研究
論文名稱(外文):Study of the Binding Domain of Hepatitis B Virus e Antigen on Neutrophils
指導教授:胡承波
指導教授(外文):Cheng-Po Hu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
中文關鍵詞:B型肝炎病毒e抗原
外文關鍵詞:Hepatitis B VirusHBeAg
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B型肝炎病毒的感染可導致急性或慢性肝炎。急性肝炎病患中,約有千分之五的人會產生致命的猛爆性肝炎,而慢性肝炎則與肝硬化和肝癌的發生有強烈的關聯。B型肝炎病毒e抗原是一種分泌性的病毒蛋白質,它不是B型肝炎病毒的結構蛋白,也不直接參與病毒的感染及複製。它的功能至今不明,但可能扮演一些免疫調節的角色,藉以幫助病毒逃離宿主的免疫反應。本實驗室過去發現e抗原能與人類周邊血液的單核白血球、嗜中性白血球及B淋巴細胞結合。為了瞭解B型肝炎病毒e抗原結合至嗜中性白血球之序列,分別構築了包含S、His及Trx三種蛋白質標籤(Tag)的4個包含N端的e抗原蛋白質片段及4個包含C端的e抗原蛋白質片段: N128 (a.a.-10 ~ 128), N112 (a.a.-10 ~ 112), N78 (a.a.-10 ~ 78), N41 (a.a.-10 ~ 41), 41C (a.a.41 ~ 149), 78C (a.a.78 ~ 149), 112C (a.a.112 ~ 149), 128C (a.a.128 ~ 149),以進行蛋白質結合部位的研究。
為了進行e抗原結合的實驗,我們首先利用西方點墨法與胜肽矩陣法分析了7種單株抗體,找出它們辨認e抗原的抗原決定位(epitope)。我們發現第一號抗體可以辨認e抗原C端第131至第139個胺基酸 AYRPPNAPI,而第三號抗體可以辨認e抗原N端第-3至第9個胺基酸 LWGMDIDPYKEF。因此,第一號抗體可用於偵測包含C端的e抗原蛋白質片段,而第三號抗體則可用於偵測包含N端的e抗原蛋白質片段。
利用流式細胞分析儀進行e抗原結合實驗時,我們發現C端的e抗原片段中,只有128C (a.a.128 ~ 149) 無法結合至嗜中性白血球,顯示出 112 ~ 128 這一段胺基酸序列可能是e抗原與細胞結合的區域。然而,我們也同時發現所有N端的e抗原片段也可結合至細胞上,因此推測在-10至112的序列中可能還有與e抗原結合的區域。我們又將N78與78C兩個e抗原片段去除蛋白質標籤後,再進行結合試驗,結果發現N78與78C都會結合至嗜中性白血球上。因此,從我們初步的結果看來,e抗原與嗜中性白血球的結合可能不只一個區域,確切的部位尚需進一步的分析。
Hepatitis B virus (HBV) causes acute and chronic infections in the liver. The acute infection may result in serious illness, and approximately 0.5% of the patients exhibit fulminant hepatitis. The chronic infection is strongly associated with the liver cirrhosis and the hepatocellular carcinoma. HBeAg, a secretory product of HBV, is neither a structural component of HBV, nor is required for the viral infectivity and replication. It is suggested that HBeAg plays an immunomodulatory role to help virus escape the host immune system.
We previously found that HBeAg could bind to human monocytes, neutrophils, and B cells. In order to identify the binding domain(s) of HBeAg to human neutrophils, four N-terminal and four C-terminal fragments of HBeAg with S, His, and Trx tags were constructed. They were N128 (a.a. -10 ~ 128), N112 (a.a. -10 ~ 112), N78 (a.a. -10 ~ 78), N41 (a.a. -10 ~ 41), 41C (a.a. 41 ~ 149), 78C (a.a. 78 ~ 149), 112C (a.a. 112 ~ 149), and 128C (a.a. 128 ~ 149).
To identify the binding of HBeAg fragment(s) on neutrophils, a panel of monoclonal antibodies (mAb) against HBeAg was first characterized using Western blotting and peptide array. We found that the epitope recognized by #1 mAb located in the sequence AYRPPNAPI (131 ~ 139 a.a.) at the C terminus of HBeAg, while that recognized by #3 mAb located in the sequence LWGMDIDPYKEF (-3 ~ 9 a.a.) at the N terminus of HBeAg. Thus, #1 mAb can be used for the detection of C-terminal fragments, while #3 mAb can be used for the detection of N-terminal fragments.
Using a binding assay followed by flow cytometry, we found that all C-terminal fragments except 128C could bind to neutrophils. These results suggest that the region of a.a. 112 ~ 128 seems to be required for the HBeAg binding. However, all the N-terminal fragments also bind to neutrophils. Thus, other sequences within a.a. -10 to 112 may also participate in the binding. We further removed the tags from N78 and 78C fragments using enterokinase. We found that both the N78 and 78C fragments without tags bound to neutrophils. Thus, our preliminary results suggest that more than one region in the HBeAg may be involved in the binding on neutrophils. The exact sequences for the binding need further investigation.
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