以EGFP為報導基因建構登革熱二型病毒PL046株之結構/非結構基因表現載體

Using EGFP as reporter for the construction of expression vectors carrying structural/non-structural genes from dengue virus type II strain PL046

洪禎憶

指導教授 ： 楊昀良

國立交通大學/生物科技學院/生物科技學系暨研究所/碩士(2011年)

https://doi.org/10.6842/NCTU.2011.01103

摘要

登革熱病毒是屬於黃質病毒科黃質病毒屬，基因全長10.7 kb，是一條正股的單股 RNA，可以產生一個巨蛋白，並經由酵素切割成三個結構與七個非結構蛋白。
在本論文所呈現的研究中，我嘗試建構能表現登革熱病毒二型 PL046 株不同基因體區段的質體。希望能取得單一區段所產生的基因產物。為了能偵測到登革熱病毒的組裝訊號，首先建構了能表現 EGFP 的兩個質體，其上分別帶有登革熱病毒的結構及非結構基因，目的在藉 EGFP 的表現提升觀察表現的靈敏度。第二、為了能大量表現 E protein，將登革熱病毒二型 PL046 株之 E gene 建構於pLP質體上，目的在單獨產生 E protein，做功能性研究。以實驗室前人的結果為基礎，我建構完成了上列質體。以定序及限制酶反應進行分析，確認這些 constructs 都沒有 non-sense mutations。

關鍵字

登革熱病毒 ； 螢光蛋白

並列摘要

Dengue virus is a single-stranded, positive-sense RNA virus with 10.7 kb genome , which can stimultaneously serves as an mRNA for translation of the viral proteins which will be digested by enzymes into three structural(C,prM,E) and seven nonstructural proteins(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5).
In this study , I constructed plasmids which contained the structural and nonstructural genes separately along with an EGFP as the reporter, for detecting the packaging signal. Next, I sub-cloned a full-length E gene from Dengue virus type II PL046 strain into pLP vector to express the E proteins for functional study. These constructs were assessed by restriction digestion and sequencing. No non-sense mutation was detected in the coding regions.

並列關鍵字

Dengue virus ； EGFP

參考文獻

Tracey J. Harvey, Wen Jun Liu, Xiang, Ju Wang, Richard Linedale, Michael Jacobs, Andrew Davidson, Thuy T. T. Le, Itaru Anraku, Andreas Suhrbier, Pei-Yong Shi, and Alexander A. Khromykh, J Virol 2004, 78(1):531-538.

邱美惠, 2007, 交大博士論文

Center for Disease Control, Taiwan (CDC, Taiwan).

Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J.-K. Vesicular Stomatitis Virus Glycoprotein Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA1993, 90, 8033-8037

Chambers TJ, Hahn CS, Galler R, et al. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 1990;44:649–688.