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標題: | Cystatin-A與C型肝炎病毒非結構性蛋白質NS3交互作用之探討 Studies on the interaction between cystatin-A and the NS3 protein of hepatitis C virus |
作者: | Wei-Chien Ma 馬瑋健 |
指導教授: | 張 鑫(Shin C. Chang) |
關鍵字: | C型肝炎病毒,非結構性蛋白質2,非結構性蛋白質3,半胱胺酸蛋白?,絲胺酸蛋白?,半胱胺酸蛋白?抑制蛋白, Hepatitis C virus,HCV,NS2/3,NS3,cysteine protease,serine protease,cystatin,cystatin-A,replication, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | C型肝炎病毒具有兩個蛋白酶,NS2/3半胱胺酸蛋白酶(cysteine protease)及NS3絲胺酸蛋白酶(serine protease)。兩者共同參與病毒多蛋白質前驅物的切割,以產生具功能性的非結構性蛋白質,協助病毒RNA複製。我們實驗室先前研究也發現基因型1b之病毒NS3蛋白質在輔助因子NS4A存在下可在第369、402與462的胺基酸位點進行自我內部截切,截切產物NS3(1-402)較NS3全長具有更高的細胞轉型能力。當利用親和性管柱分析與NS3(1-402)會形成複合物的細胞因子時,發現Huh7細胞中的cystatin-A可與NS3(1-402)被共同純化出來,表示兩者間可能具有交互作用。Cystatin-A為一半胱胺酸蛋白酶抑制蛋白(cysteine protease inhibitor),可調控細胞中半胱胺酸蛋白酶的活性,參與表皮的發育及維護。當細胞中cystatin-A的表現量下降時,可能造成半胱胺酸蛋白酶活性不受調控而導致細胞癌變。本研究在探討cystatin-A半胱胺酸蛋白酶抑制蛋白與NS3(1-402)被共同純化出來的生物意義,以及此蛋白酶抑制蛋白是否會抑制NS2/3的自我切割能力。結果發現在293T細胞中,cystatin-A雖會與NS3全長有交互作用,但對NS3的表現量和蛋白酶活性並無顯著影響,cystatin-A也不會抑制NS2/3的蛋白酶切割活性。進一步以即時定量聚合酶連鎖反應探討cystatin-A是否影響病毒之複製,結果顯示cystatin-A蛋白質的表現,會減少病毒基因體RNA的複製,其調節機制有待進一步探討。 Hepatitis C virus (HCV) possesses two viral proteases, NS2/3 cysteine protease and NS3 serine protease, which participate in the viral polyprotein processing to generate functional non-structural proteins. In addition to polyprotein processing, our previous studies also demonstrated an internal NS3 cleavage activity of HCV genotype 1b that occurred at residue 369, 402 and 462 in the presence of NS4A. The internal cleavage product, NS3(1-402), had a higher transforming activity than the full-length NS3. By performing tandem affinity purification, cystatin-A was co-purified with NS3(1-402) protein, implying an association of the two proteins. Cystatin-A, as a cysteine protease inhibitor, can modulate cellular cysteine protease activity and plays roles in epidermal development and maintainance. The down-regulation of cystatin-A was suggested to be involved in tumorigenesis, including cancers of breast, pancrea, brain, and lung. In this study, biological significances for the association between cystatin-A and NS3(1-402) were examined. Knowing that HCV NS2/3 possesses cysteine protease activity, a potential inhibitory effect of cystatin-A was also examined. The results showed that cystatin-A had no inhibitory effect to the NS2/3 cysteine protease and NS3 serine protease activity. On the other hand, cystatin-A inhibited the RNA replication of HCV JFH-1. Molecular mechanisms involved in the inhibition of HCV RNA replication remain to be elucidated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58237 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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