檢測黑女王細胞病毒的IRES 與其它IRES 在不同昆蟲細胞株中之轉譯活性

Translational activity of Black queen cell virus’ IRES and other IRESs in different insect cell lines

周易德(Yi-De Chou)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6840/cycu201400702

摘要

(I) 桿狀病毒表現系統 (Baculovirus expression vector system; BEVS) 為目前廣泛使用的真核表現系統之一，為達生產蛋白複合物或多價疫苗之目的，本實驗室致力於開發桿狀病毒表現系統的多效表現載體，以期能同時表現兩種以上之蛋白質。而在昆蟲細胞中具有轉譯活性之核醣體內轉子 (Internal Ribosomal Entry Site, IRES) 為實驗室開發多效表現載體之重要元件。IRES為一段RNA序列，其所形成之特殊二級結構能吸引轉譯起始因子，因而驅動下游的開放閱讀框架 (oper reading frame; ORF) 進行轉譯起始作用。在昆蟲的RNA病毒中，常具有5’端非轉譯區 (5’ untranslation region; 5’UTR) 及兩開放閱讀框架間的區域 (intergenic region; IGR)，皆已被證明具有IRES之活性，並在病毒複製上扮演重要的角色。本研究中利用新IRES二級結構比對與搜尋工具 (VIPS)，預測黑女王細胞病毒 (Black queen cell virus; BQCV) 中的IGR具有IRES活性，但5’UTR則不具有IRES活性。為證實VIPS的準確度，我們將此兩段序列構築至含紅、綠螢光蛋白基因之雙效表現載體中，分析紅、與綠螢光蛋白表現，以評估IRES在桿狀病毒表現系統中之活性。另外我們也同時比較來自常蚜病毒 (Rhopalo- siphumpadi virus; RhPV)、蟋蟀麻痺病毒 (Cricket paralysis virus; CrPV)、榕樹透翅毒蛾病毒 (Perina nuda virus; PnV)、傳染性軟化病毒 (Infectious flacherie virus; IFV) 以及幼蜂皺縮病毒 (Sacbrood virus; SBV) 等昆蟲病毒之IRESs在秋行蟲軍卵巢細胞株 (Sf9、Sf21) 及擬尺蠖夜蛾幼蟲細胞株 (High-Five) 中之轉譯活性。以螢光顯微鏡、螢光偵測儀及西方墨點法分析螢光蛋白表現，發現BQCV-IGR之IRES活性略高於5’UTR ，顯示VIPS預測之正確性。PnV和RhPV的IRESs則在各細胞株中具有高轉譯活性。其中在Sf9及Sf21細胞株中，PnV IRES活性較RhPV IRES略高，但在High-Five細胞中則反之。進一步以RNA結構預測軟體分析各IRESs的二級結構時，找出可能具有強轉譯活性之結構區域並將其結合，最後提出一個新穎的人工嵌合之IRES序列。

(II) A26蛋白是牛痘病毒之胞內成熟病毒 (intracellular mature virus；IMV) 的鞘膜蛋白質，而同為鞘膜蛋白的A27蛋白質會與A26利用雙硫鍵形成蛋白質複合物，A26蛋白與牛痘病毒進入細胞的機制息息相關，而A27亦為產生中和抗體之重要抗原。為透過解開A26-A27分子間結構之關聯性，進而了解痘病毒之致病機轉，並用以開發抗痘病毒藥物與疫苗等應用。我們利用實驗室所開發之桿狀病毒多效表現載體，同時表現A26和A27蛋白質。以核多角體基因啟動子驅動A26蛋白表現，而由RhPV IRES介導A27蛋白表現。在西方墨點法的分析下，我們能成功的共表現A26和A27蛋白質，而在非還原條件下，也能觀察到A26與A27形成之複合物。當進一步利用Flag Tag純化套組進行A26蛋白純化時，我們能成功地從單表現A26蛋白之重組病毒感染的細胞萃取液中純化到A26蛋白。然而在A26及A27蛋白同時表現之細胞萃取液中，卻無法有效同時純化A26及A27之蛋白複合物，顯示A26-A27蛋白複合物形成時，可能會影響Flag Tag與抗體之間的辨認。未來希望能大量純化A26蛋白或A26-A27之蛋白複合物進行結構性分析，以期應用於病毒致病機轉或疫苗開發等研究。

關鍵字

桿狀病毒表現系統 ； 核醣體內轉子 ； 黑女王細胞病毒 ； 牛痘病毒 ； A27 ； A26

並列摘要

(I) Baculovirus expression vector system (BEVS) is currently widespread use of exogenous protein eukaryotic expression systems. To produce the protein complexes or polyvalent vaccine, our laborary dedicates to develop the polycistronic expression vector in BEVS to simultaneously express two or more proteins. Internal Ribosomal Entry Sites (IRESs) is an RNA sequence that forms specialized secondary structure and can attract translation initiation factors to mediate downstream open reading frame (ORF) translation without traditional cap-recognition. IRES derived from insect RNA virusis usually located on the 5’-Untraslated region (5’-UTR) or intergenic region (IGR) of viral genome. The region is proved to have IRES acivitity and play important roles on virus replication. In this study, we used IRES search system, named VIPS, to explore new potential IRES candidates. Here, the IGR of Black Queen Cell Virus (BQCV), instand of 5’-UTR, may have IRES activity predicted by VIPS. To confirm the accuracy of VIPS program, the BQCV IRES of 5’-UTR and IGR were cloned into IRES-based bi-cistronic reporter system to evaluate the IRES activity in BEVS, which polyhedrin promoter drives the DsRed expression and IRESs derived from different insect RNA virus mediate the EGFP expression. In addition, we also examined the translational activities of six IRESs from Rhopalosiphum padi virus (RhPV), Cricket Paralysis Virus (CrPV), Perina nuda Virus (PnV), Black Queen Cell Virus (BQCV), Infectious Flacherie Virus (IFV) and Sacbrood Virus (SBV) in Spodoptera frugiperda cell line (Sf21，Sf9) and Trichoplusia ni cell line (High-Five). By fluorescence microscope and western blot analysis, we firstly fould the EGFP expression mediated by BQCV (IGR) IRES was slightly higher than BQCV (5’UTR) IRES. When further comparing the different IRES, we observed that the EGFP expression mediated by PnV and RhPV IRESs were higher than all others among three cell lines. To quantitatively analyze the IRES activity, we measured the intensity of EGFP by fluorescence spectrophotometer under the DsRed normalization. The data showed that in Sf21 or Sf9 cells, the activity of PnV IRES was higher than RhPV IRES but it was contrary in High-Five cells. Finally, through analyzing the RNA secondary structures of these IRESs by M-fold software, we proposed some loops were crucial for strong IRES activity in insect cells. Combining these regions, we propose a novel chimeric IRES model.

(II) Vaccinia virus A26 protrin is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. The A27 protein formed a protein complex with A26 protein in infected cells as well as in IMV. Presence of A26 protein is closely related to vaccinia virus entry into host cell’s mechanisms and A27 protein is also an important antigen for neutralizing antibody. To further discover the deadly mechanism of vaccinia virus and apply to the development of anti-pox drugs and vaccines through the understanding of the intermolecular structures of A26-A27 protein complex, we use our developed polycistronic expression vector in baculovirus expression system to co-express the A26 and A27 protein. In this study, the DNA fragments of open reading frames of A26 and A27 genes were cloned into polycistronic vector that polyhedrin promoter drives the A26 expression and RhPV IRES mediates the A27 expression. We successfully expressed the A26 and A27 proteins by western blotting analysis. In addition, we also detected the formation of A26-A27 protein complexes under non-reducing condition. When further purifying the A26 protein by flag tag purification kit, we purified the A26 protein from the cell lysate infected by A26-expressing virus. However, it could not efficiently purify the A26-A27 complexes from cell lysates infected by A26-A27 co-expressing virus. It might imply that the formation of A26-A27 complexes impeded the flag tag recognition by anti-flag antibody. We hope to produce and purify the A26 protein or A26-A27 complexes for structure analysis in the future and then help the investigation of viral pathogenesis and vaccine development.

並列關鍵字

A26 ； A27 ； Baculovirus expression vector system ； Black queen cell virus ； Vaccinia Virus ； Internal Ribosomal Entry Site

參考文獻

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