使用FISH-PCR尋找D型肝炎病毒抗原所誘導的基因

Investigation of HDV antigen induced genes by FISH-PCR

張瑞玶(Jui-Pin Chang)

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)之基因體是由一條1.7 kb的環狀單股RNA所構成。HDAg為HDV唯一產生的蛋白質，由HDV反基因股的一個特定ORF轉譯而出。HDAg可分為兩種不同的種類：(一) S-HDAg, 分子量為24–kDa (195個胺基酸)主要功能為參與HDV RNA之複製；(二) L-HDAg, 分子量為27-kDa（214個胺基酸），其功能為參與HDV病毒顆粒的包裝並且與抑制HDV RNA複製有關。本篇實驗目的，為了尋找在HDV複製時與之相關的蛋白如轉錄因子、細胞蛋白、或是與HDAg本身功能相關的蛋白…等等，在此利用FISH PCR技術，在穩定表現S-HDAg或L-HDAg的人類肝癌細胞株，與不表達S-HDAg或L-HDAg的人類肝癌細胞株中，篩選出具有差異性表現的基因。然後將受S-HDAg或L-HDAg誘導的基因經過菌落PCR、酵素切割的分析、定序及序列比對後，挑選兩個基因(C, E)做後續研究。亦即利用半定量PCR及北方墨點法確認這兩個基因增加的可信度，結果顯示C基因mRNA表現量與L-HDAg表現量的成正相關；而E基因只利用半定量PCR做初步確認，結果顯示E基因只受到S-HDAg的調控進而增加mRNA的表現。本實驗初步鑑定出會受HDAg影響所誘導的基因有C和E，至於兩者具有什麼相關性且在HDV的複製過程中扮演何種角色，則有待後續實驗證明之。

關鍵字

D型肝炎病毒

並列摘要

The genome of hepatitis delta virus (HDV) is composed of a circular single-stranded RNA molecule of 1.7-kb that is the smallest, and the only known circular RNA genome of the aminals RNA virus. The HDV 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), essential for particle assembly and inhibition of HDV RNA replication. The aim of this project is to identify cellular genes that are related to HDV replication by HDAgs induction. Therefore, we used FISH PCR technology to screen differentially expressed genes among Huh7 cells that stably expressing S-HDAg or L-HDAg. The differential expressed genes, which are induced by S-HDAg or L-HDAg were cloned by PCR and verified by autosequence. We select two genes, named C and E genes for further study. To confirm these two genes are truly HDAgs induced genes, semi-quantitative and Northern blotting are employed to quantify the expression levels of these genes. Our results indicated that the mRNA level of C gene correlated with L-HDAg expression level. Analyzed by RT-PCR, the mRNA level of E gene and is induced only by S-HDAg. Overall, we identified that both C and E genes are induced by HDAg. How the genes interact with HDAg and what role these two genes play in HDV replication need to be proved in the future.

並列關鍵字

HDV

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