- 學位論文
探討D型肝炎病毒抗原之啟動子的專一性
Study the promoter specificity of Hepatitis D Virus antigen
摘要
在自然狀態下，D型肝炎病毒(Hepatitis Delta Virus，簡稱HDV)只存在於共同感染B型肝炎病毒(Hepatitis B Virus，簡稱HBV)的病人中。HDV由一條1.7 kb的環狀單股RNA所構成，而且HDV是最小且環狀的RNA動物病毒。在一般狀態下，其基因體有70%的鹼基對互補可形成雙股RNA的桿狀結構；在HDV的複製週期中，必須利用HBV所產生的蛋白(HBsAg)當莢膜，才能組裝完整的病毒顆粒。 由先前的研究結果顯示，在HDV的cDNA序列中，不論是基因股或是反基因股都有promoter的活性存在，並且證實於反基因股中有一段29個nt的區域(相對於HDV-1.9的第999~1028個nt)，具有內生性promoter的活性；至於基因股之promoter活性的探討，則缺乏有利的證據加以證實與定量。根據本實驗室之前的研究指出，HDV的cDNA反基因股970~1070 nt（promoter-8）為HDV之內生性promoter，且其正反向序列皆具有promoter的活性。本篇實驗即以此正反向HDV promoter-8序列進行研究，利用產生的luciferase及Green Fluorescent Protein (GFP)及Hepatitis delta small antigen（SmAg）作為reporter基因，使用RT-PCR的方法比較promoter-8轉錄能力的差異，初步判定當promoter-8表達自己的基因SmAg時，具有選擇性表現，再以Western blotting的方式，同樣得到驗證。最後，利用更靈敏的免疫螢光染色（Fluorescence stain）與GFP做比較，並於顯微鏡下觀察螢光的表現，進而證實promoter-8改變方向後，對表達SmAg之活性亦隨之改變。總結言之，本實驗說明了HDV的cDNA的反基因股上，promoter-8序列確實對其本身的基因，具有選擇性表現，在promoter的研究中可能是新的發現，在未來或許可以利用此promoter序列，對研究HDV RNA複製及轉譯的機制、基因本身可能具有選擇性表現及細胞內可能存在掃瞄基因蛋白等研究，提供一個新的詮釋。
關鍵字
專一性表現並列摘要
In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). The genome of HDV is composed of a circular single-stranded RNA molecule of 1.7-kb that is the smallest, and only known circular RNA genome of the aminals RNA virus. Sequence analysis of the HDV genome revealed that as many as 70% of nucleotides have to undergo intramolecular base pairing, thus allowing the RNA to fold into a rod-like structure. HDV RNA genome are assembled using the envelope protein (HBsAg) of HBV. From previously study indicated that HDV cDNA contains endogenous promoters both in genomic and antigenomic strands. It has been shown that the antigenomic cDNA promoter-like sequence of HDV was located to a 29-nucleotide region (corresponding to HDV-1.9 nucleotides 999 ~ 1028). Whereas the promoter activity of the genomic HDV cDNA was still unclear. In ours previously study, we known that anti-genomic HDV的cDNA has an endogenous promoter activity and is located in nucleotide 970 ~ 1070（promoter-8）. The activity of this promoter is not restricted by its orientation. In this study, we use luciferase and Green Fluorescent Protein (GFP) as reporter genes that compare with HDV SmAg gene to determine the promoter-8 activity. The results of RT-PCR show this promoter-8 has specific-orientation activity for SmAg. We also use western blotting to comfirm this result. Furthermore, we use Immunofluorescence stain to demonstrate the difference by staining HDV SmAg. Taken together, we propose that promoter-8 has SmAg specific expression activity. These results may have implications for studying HDV RNA replication and transcription mechanisms.