常蚜病毒5’末端序列在哺乳動物細胞和昆蟲細胞中轉錄和轉譯活性之分析

Transcription and translation activity of Rhopalosiphum padi virus 5' UTR inter ribosome entry site in mammalian and in insect cell

吳奕珍(I-Chen Wu)

指導教授 ： 吳宗遠

中原大學/理學院/化學研究所/碩士(2006年)

https://doi.org/10.6840/cycu200600417

摘要

無資料

關鍵字

promter ； RhPV IRES

並列摘要

Rhopalosiphum padi virus (RhPV) is an insect virus of the family Dicistrviridae. We had demonstrated that the 579-nucleotide long 5’ untranslated region (5’UTR) of RhPV internal ribosome entry site (IRES) can mediate cap-independent translation efficiently in baculovirus infected insect cells. Recently, this RhPV IRES was showed to be a cross-kingdom IRES that can function efficiently not only in insect cells but also in mammalian and plant in vitro translation system. To substantiate the in virto observation, a cytomegalovirus (CMV) promoter based bi-cistronic mammalian cell expression vector was constructed. Transiently transfection assay showed that the efficacy of RhPV 5’UTR IRES mediated translation in CHO was higher than in COS-1, and HeLa cells. For the recombinant baculoviruses with CMV promoter can mediated gene transduction in mammalian cells, we generated a recombinant virus, vAcCMVD-Rhir-E. As vAcCMVD-Rhir-E transduced COS-1, it was consisted with pCMVD-Rhir -E plasmid transfection assay, the transduced COS-1 cells revealed the red fluorescence as well as green fluorescence. However, as the vAcCMVD- Rhir-E infected insectSf21 cells, only the second cistron green fluorescence was observed. Northern blot showed that the major mRNA species hybridized with the EGFP gene fragment probed was the 0.8 kbp EGFP mRNA. This may implied the bi-cistron mRNA was post transcriptionally spiced or the RhPV 5’UTR IRES contains an internal promoter. To clarify this puzzle, a promoterless recombinant baculovirus, vAc∆CMVD-Rhir-E, was constructed. Interestingly, the vAc∆CMVD-Rhir-E infected insectSf21 cells still revealed the green fluorescence. These results indicated the RhPV 5’UTR IRES contains a cryptic promoter in baculovirus infected insect cells. This is the first finding that an IRES sequence can function as an internal ribosome entry structure as well as a promoter.

並列關鍵字

RhPV核酸內轉譯子 ； 啟動子

參考文獻

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