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  • 學位論文

C型肝炎病毒包膜蛋白的特性及引發免疫反應能力之探討

Characterization and Immunogenicity of Hepatitis C Virus Envelope Proteins

指導教授 : 翁啟惠

摘要


全世界大約有百分之三的人口感染C型肝炎病毒(HCV)。服用Ribavirin並同時注射聚乙烯二醇化甲型干擾素(pegylated interferon-α)是目前的治療方法,但是此療法對不同基因型C型肝炎病毒的感染有不同效果。然而現今仍然沒有可對抗或預防C型肝炎病毒的疫苗,因此如何製備出有效的疫苗是一大課題。 C型肝炎病毒的基因組可轉譯出一條多蛋白(polyprotein),並且能被宿主以及病毒本身的蛋白酶切割成多段結構蛋白以及非結構蛋白。其中,包膜蛋白(envelope protein)屬於結構蛋白,表現在病毒的表面上,包含了E1及E2兩個蛋白,並且在結合受體(receptor)與病毒進入細胞上扮演了重要的角色。E1和E2蛋白是高度醣化(glycosylated)的醣蛋白,蛋白質的醣化對於蛋白質折疊以及受體的結合來說都十分重要,不過醣也可以遮蔽蛋白質上重要的、具有功能性的位置,讓宿主無法對病毒產生有效的抗體來對抗病毒的感染。 我的研究是探討醣在C型肝炎病毒包膜蛋白E1以及E2醣蛋白的角色。我先從人類293T細胞中製造且純化出全長(full-length)的E1E2膜蛋白,以及去除部分C端(C-terminal truncated)的E2蛋白。接著,再利用醣內切酶 H (Endoglycosidase H, Endo H)酵素去除這兩種蛋白上除了第一個乙醯葡萄糖胺(N-acetylglucosamine)之外大部分的醣,這樣處理後的蛋白即為單醣形式(mono-glycosylated form),而沒有用醣內切酶 H處理過的包膜蛋白,稱之為全醣形式(fully-glycosylated form)。接著我比較了這兩種不同醣形式的全長E1E2以及C端部分切除的E2蛋白上的醣化程度及種類,確定醣是否切除的完整;並用圓二色光譜(circular dichroism)比較其二級結構,同時評估其對受體CD81的結合能力,探討切除了醣的蛋白是否仍然具有一樣的結構及活性。最後,並試圖探討C型肝炎病毒包膜醣蛋白引發老鼠免疫反應的能力。 實驗結果發現,單醣形式上有偵測到的醣化位置都只剩下一顆乙醯葡萄糖胺,而二級結構與全醣形式相似,與受體CD81結合能力則是明顯的比全醣形式來的高。另外,在引發免疫反應的部分,只有全醣形式可以引發出較高的抗體效價,而單醣形式的效果比較差。

並列摘要


It was estimated that about 3% of the worldwide population were infected with hepatitis C virus (HCV). The current therapy is ribavirin combined with pegylated interferon-α, and the effect was differential for infections with various HCV genotypes. Until now there is still no vaccine available. The genome of HCV encodes a polyprotein which could be cleaved into structural and nonstructural proteins by host and viral proteases. The envelope proteins are structural proteins which are expressed on the viral surface and are comprised of heterodimers of E1 and E2. They play an important role in receptor binding and virus entry. HCV E1 and E2 proteins are highly glycosylated. Glycans may be crucial for protein folding or receptor binding or both. Glycans also can mask the functionally important region of protein so that proteins may not produce effective antibodies to defeat the viruses. Our ultimate goal is to investigate the roles of glycans on HCV E1/E2 glycoproteins, especially on the immunogenicity. Recombinant HCV envelope proteins were produced from human 293T cells. The purified proteins are either used directly (fully-glycosylated) or digested with Endoglycosidase H to remove all the glycans except the first GlcNAc (mono-glycosylated). The glycosylation patterns of fully- and mono-glycosylated HCV envelope proteins were further examined by glycan profiling. Their structural and functional properties were also evaluated with circular dichroism spectroscopy and with binding activity to CD81, a receptor of HCV. In addition, the immunogenicity of HCV envelope proteins in various forms was investigated. The results showed that the N-glycans of mono-glycosylated proteins were mostly one HexNAc, and the secondary structures of fully- and mono-glycosylated proteins were similar. Furthermore, the CD81 binding activity of mono-glycosylated proteins was higher than fully-glycosylated proteins. However, the immunogenicity of fully-glycosylated proteins was higher than mono-glycosylated HCV envelope proteins.

並列關鍵字

HCV envelope protein E1E2 glycosylation Endo H immunogenicity

參考文獻


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