研究 HDV cDNA Promoter 活性和 HDV antigen之關係

Study the association of HDV antigen and HDV promoter

陳德合(Te-Ho Chen)

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 ) 由一條1.7 kb的環狀單股RNA所構成。HDAg是經由HDV之中唯一的 Open Reading Frame ( ORF ) 所轉譯的蛋白，可分為兩種不同的類型， Small-HDAg ( S-HDAg ) 其分子量約為24 KDa ( 195個胺基酸 )，主要功能為參與 HDV RNA的複製；Large-HDAg ( L-HDAg ) 其為27 KDa ( 214胺基酸 )大小，其功能為參與HDV病毒顆粒的包裝。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個核苷酸( nt )的區域，具有內生性promoter的活性；至於基因股之promoter活性的探討，則尚未加以證實。根據本實驗室之前的研究指出，位於HDV cDNA反基因股的970~1070 nt（TR-P 1）為HDV之內生性promoter，且其正反向序列皆具有promoter的活性。另一個位於HDV的ORF內( TR-I 1 )，同樣的其正反向序列皆具於有promoter的活性。因此本實驗就以決定HDV cDNA promoter的最短序列進行研究，分別對TR-P 1及TR-I 1進行刪除實驗，利用luciferase reporter system的方法，來精確定量各個promoter片段的活性大小。我們發現TR-I 1隨著片段的縮短其promoter活性卻隨著增加，尤其以TR-I 5活性最強。而TR-P 1則隨著片段的前後縮短promoter活性卻隨著下降，甚至對TR-P 1進行定點突變實驗也是相同的情形。另一方面也以HDAg ( L-HDAg；S-HDAg ) 刺激 HDV cDNA promoter，來觀察promoter 活性之變化。發現L-HDAg及 S-HDAg的確會對TR-I 5 和TR-P 1 的promoter活性造成不同的影響。
總觀上述，本實驗說明在HDV 的cDNA序列之上，的確含promoter的活性，並顯示這兩段的promoter序列，分別位於HDAg 的ORF內區與外區，在未來或許可以利用此兩段promoter序列，對於研究HDV RNA複製及轉譯的機制提供一個新的詮釋。

關鍵字

啟動子

並列摘要

The genome of hepatitis delta virus ( HDV ) is composed of a circular single-stranded RNA molecule of 1.7 Kb. The gene of hepatitis delta antigen ( HDAg ) consists of two related proteins. The small form is a 24 KDa ( 195 amino acids ) of S-HDAg, which is essential for replication of HDV RNA genome. The large form is L-HDAg, which is a 27 KDa ( 214 amino acids ), that is essential for particle assembly of HDV RNA. It has been indicated that HDV cDNA contain endogenous promoters both in genomic and antigenomic strands. The position of the antigenomic HDV cDNA promoter-like sequence was located to a 29-nucleotide region, whereas the promoter activity of the genomic HDV cDNA was still unclear. We already knew that antigenomic HDV cDNA has an endogenous promoter activity and is located in TR-P1 fragment, the activity of this promoter is not restricted by its orientation. Another promoter is located in TR-I 1 fragment. In this study, we attempt to identify the essentral promoter sequence of these two HDV cDNA fragments. First, HDV cDNA fragments were inserted into PGL-3-basic vector, and using luciferase as a reporter gene to indentify the HDV cDNA promoter activity. We found that TR-I 5 activity was higher than TR-I 1.The deletion and point mutation analysis in TR-P 1 provided further information about TR-P 1 promoter activity. Finally, we cotransfected L-HDAg or S-HDAg to study HDAg effects on HDV cDNA promoter activity. We observed that L-HDAg and S-HDAg can alter the specific HDV cDNA promoter activity.

Overall, the HDV cDNA sequences containing promoter activity were found and the novel promoter sequence may be used for studying HDV RNA replication or transcription mechanism in the future.

並列關鍵字

promoter

參考文獻

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