在大腸桿菌大量表現與純化B型肝炎病毒重組X蛋白之反式活化區

Expression and purification of recombinant transactivation domain of hepatitis B virus (HBV) X protein in Escherichia coli

周翰林(Han-Lin Jou)

指導教授 ： 林意然

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2010年)

摘要

根據之前報告，估計全球感染慢性B型肝炎病毒的人口已超過3.5億，特別是在亞洲地區相當盛行。而且感染B型肝炎病毒將會引起肝炎、肝硬化、肝癌疾病。肝癌的產生普遍發現在感染慢性B型肝炎病毒及C型肝炎病毒的病人身上，但對於B型肝炎病毒感染與肝癌之間發生的分子機制，到現在還不是很清楚。
B型肝炎病毒，屬於Hepadnaviride的家族中最小的DNA病毒，DNA基因長度為3.2kb，並且為一環狀部分雙股的結構。感染B型肝炎病毒宿主為人類。DNA基因上共有四個開放譯讀區，分別是S、P、C、X譯讀區。我們實驗室主要感興趣於HBx蛋白的反式活化區。B型肝炎病毒X蛋白（HBx）是一個多功能性的蛋白，之前文獻指出HBx 蛋白質跟調控細胞基因表現與病毒複製有關，包括活化轉錄作用、細胞凋亡作用、磷酸化與乙醯化作用、與修復受損DNA蛋白質結合進而刺激病毒複製等。
HBx 蛋白質是B型肝炎病毒四個開放譯讀區最小的譯讀區，由154個胺基酸組成，編譯出片段長度約17.5 kDa。在HBx蛋白上分成調控區（regulatory domain）、轉錄活化區（transactivation domain）兩大部份。在大部分被感染B型肝炎病毒動物的HBx蛋白上，發現具有很高的保留性，但仍缺乏已知同源性蛋白的特性，並且尚未以核磁共振或X光晶體繞射解出HBx 蛋白質的三度空間立體結構。
值得一提的是市面上已有許多抗B型肝炎的藥，例如：α-干擾素，或是核苷類似物，但近來文獻指出這些藥物具有副作用與時效性短的問題，因此，這些說明了發展出抗B型肝炎新藥的必要性，並進一步的指出B型肝炎病毒HBx蛋白質三度空間結構的重要性，以便能夠了解HBx蛋白的反式活化區對於B型肝炎病毒感染與肝癌之間發生的分子機制，並以此蛋白為藥物標靶，進而設計出更有效的藥物。
目前實驗室已建構重組HBx蛋白的反式活化區表現系統，經誘導劑誘導後可大量表現出HBx融合蛋白質，並利用親合性管柱層析技術，純化得到相當純度的融合蛋白質。

關鍵字

B型肝炎病毒

並列摘要

It was estimated that more than 350 million individuals were infected with chronic hepatitis B virus (HBV). Especially, half of the world’s population lives in an area with high HBV prevalence. The HBV will eventually develop severe liver diseases, including liver cirrhosis, and hepatocellular carcinoma (HCC), one of the most common forms of human cancer. But its involvement in the development of HCC is still unknown.
HBV is a prototype of Hepadnaviridae, a small hepatotropic DNA viruse with partially double-stranded DNA genome. The genome structure of HBV is a 3.2-kb DNA. Natural host for HBV is humans. The ORFs correspond to core protein, envelope protein (surface antigen), polymerase (pol protein), and HBx protein. Our group is interested in transactivation domain of Hepatitis B Virus x protein (HBx). The HBx protein is a multifunctional protein. It has been suggested to affect viral replication by modulating a wide variety of cellular processes, including transcriptional transactivation, apoptosis, phosphorylation or acetylation, as well as interaction with damaged DNA binding proteins.
HBx is encoded by the smallest HBV ORFs and consists of 154 amino acids, with a molecular weight of about 17.5 kDa. The HBx gene is conserved among all mammalian hepadnaviruses. But the X protein lacks homology with known proteins. And lacks three-dimensional structure determined by from nuclear magnetic resonance (NMR) or X-ray crystallography. Unfortunately, the other reports showed that anti-HBV drugs such as interferon alpha and nucleoside analogs have adverse reactions or effect only in the short time, Therefore, the development of novel antiviral strategies is important. The information of the three-dimensional structure of the HBx protein will provide an alternative to understand the molecular mechanism between HBV infection and HCC. Our group has constructed the recombinant HBx gene and expressed the fusioned protein. The fusioned transactivation domain of the HBx protein was obtained in a large amount from supernatant of lysate, but not from inclusion body, and purified with Ni-NTA by AKTA prime plus (GE). In the future work enzyme digestion will be proceeded by digestion enzyme and the HBx protein will be purified by Ni-NTA and AKTA purifier.

並列關鍵字

hepatitis B virus (HBV)

參考文獻

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