利用二維膠電泳分析D型肝炎抗原與宿主細胞蛋白之關係

Analysis the association between HDV antigen and host cell protein by two-dimensional gel electrophoresis

尤信超(Shin-Chao Yu)

指導教授 ： 許國堂

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2005年)

摘要

D型肝炎病毒( Hepatitis Delta Virus，簡稱HDV )，為一個負單股RNA病毒包含了一條1.7kb的環狀RNA，而且也是B型肝炎病毒( Hepatitis B Virus，簡稱HBV )的衛星病毒。正常狀態下，HDV會產生兩種蛋白(HDAg)分別是Small HDAg幫助HDV的複製；Large HDAg會抑制HDV的複製與幫助病毒顆粒的合成。
先前的研究中，我們雖然都知道HDAg的某些功能，但是對於其真正的作用點以及作用機制，目前來說並不是非常的清楚。而從本實驗室先前的研究中發現到，HDAg可以不用透過免疫沉澱的步驟，直接與Protein A sepharose有所結合作用。因此，再本篇實驗中，利用我們所做出來的Huh-7表達HDAg的stable clone (Large and Small)兩種細胞株的核蛋白，透過Protein A sepharose binding assay之後，再利用二維膠電泳來進行分析。觀察HDAg在細胞中會與哪些蛋白有所結合能力。我們分別分段比對兩組實驗中有表達以及沒表達HDAg的實驗結果，發現到不管是在Large HDAg或者是Small HDAg中， 都可以觀察到有差異點的存在。
接著就希望能將這些差異點取下來，利用GC-MASS分析之後，找出其為何種蛋白。對於以後在HDV的研究上，可以提供一些新的想法以及更多的實驗方向。

關鍵字

二維膠電泳分析

並列摘要

Hepatitis delta virus (HDV) is a negative single-strand RNA virus that contains a circular RNA genome of 1.7 kilobase and is also a subviral satellite of hepatitis B virus (HBV). HDV encodes two isoforms of delta antigen (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. Although we knew about some of the functions of HDAgs but the mechanism and interacting proteins are mostly unknow. In our laboratory, we find that HDAg can bind with Protein A sepharose without the aid from the antibody-mediated immunoprecipitation. In this study, we generated the HDAg stable expressing clone (Large and Small) from Huh-7 cells and separated the cytoplasma and nuclear protein for analysis. After Protein A sepharose binding assay. The binding proteins were analyzed by two-dimensional gel electrophoresis and determined which protein will interact with HDAg in these stable clone cell lines. We found that nuclear and cytoplasma fractions of both small and large expressing cells contain proteins that specifically associated with the HDAg isoform.
Overall, we found that after two-dimensional gel electrophoresis, unique spots were identified. We will analyze these spots by GC-MASS in the future and these will be generated protein data banks may provide a new insight to the HDV research.

並列關鍵字

two-dimensional gel electrophoresis

參考文獻

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