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標題: | Cystatin-A與C型肝炎病毒非結構性蛋白質NS3之功能性結合作用 Functional interaction between cystatin-A and hepatitis C virus non-structural protein NS3 |
作者: | Yu-Jia Su 蘇堉嘉 |
指導教授: | 張鑫 |
關鍵字: | C型肝炎病毒,非結構性蛋白質NS3,NS2-3半胱氨酸蛋白酶,Cystatin A, Hepatitis C virus,Non-structural protein NS3,NS2-3 cysteine protease,Cystatin A, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | C型肝炎病毒(Hepatitis C Virus)為一種單股正向的RNA病毒,感染宿主細胞後,可於內質網上轉譯出一條多蛋白質(polyprotein),再藉由細胞與病毒的蛋白酶切割成各病毒蛋白質。非結構性蛋白質NS3具有631個胺基酸,其internal cleavage產物NS3(1-402)具有較NS3全長更強的細胞轉型能力。本研究利用Tandem Affinity Purification的系統尋找會和NS3(1-402)有交互作用並共同被純化出來的細胞蛋白質。利用LC-Mass/Mass鑑定出其中分子量9 kDa與34 kDa會和NS3(1-402)產生交互作用的細胞蛋白質分別為cystatin A與PRSS3。利用免疫沉澱的方法更進一步確認cystatin A與全長NS3的交互作用。Cystatin A為一個cysteine protease的抑制物,在許多癌症的形成與轉移中扮演重要的角色,亦有研究指出cystatin A具有抑制病毒複製的能力。C型肝炎病毒 NS2-3蛋白質是一個cysteine protease,具有自我切割的功能。為了瞭解cystatin A是否會對NS2-3的cysteine protease activity產生影響,本研究在細胞中共同表現NS2-3 cysteine protease與cystatin A蛋白質,結果顯示NS2-3 cysteine protease的自我切割產物比例有下降的情況,推測cystatin A可能會抑制C型肝炎病毒cysteine protease的活性。Cystatin A在C型肝炎病毒增殖與致病機轉上的角色有待進一步的研究。 Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus. It encodes a polyprotein precursor at the endoplasmic reticulum of infected cells. The polyprotein precursor can further processed into structural and non-structural protein by cellular and the viral proteases. Non-structural protein 3 (NS3) consists of 631 amino acids. Our previous studies have demonstrated an internal cleavage activity of the genotype 1b NS3 protein and an enhanced transforming activity of the major cleavage product NS3(1-402) compared with the full-length NS3 protein. In this study, tandem affinity purification system was used to find out cellular factors which interact and can be co-purified with NS3(1-402). LC-Mass/Mass analysis identified the co-purified 9 kDa and 34 kDa proteins to be cystatin A and PRSS3, respectively. The interaction between cystatin A and the full-length NS3 was further confirmed by co-immunoprecipitation. Cystatin A is a cysteine protease inhibitor. It plays important roles in the development and metastasis of several types of cancer. It was also demonstrated to be involved in viral multiplication. HCV NS2-3 protein is a cysteine protease with auto-cleavage activity. To investigate whether cystatin A can inhibit NS2-3 cysteine protease activity, NS2-3 and cystatin A were coexpressed in cultured cells. Coexpressing of cystatin A with NS2-3 resulted in a decreased level of the auto-cleaved NS3 product, indicating that cystatin A may inhibit NS2-3 cysteine protease activity. The roles of cystatin A in HCV multiplication and pathogenesis need to be further investigated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62866 |
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顯示於系所單位: | 微生物學科所 |
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