臺灣博碩士論文加值系統

目前已有許多研究顯示正股RNA病毒的複製子（replicon）可以用來研究病毒的複製，當作表現外源基因的系統，或發展為篩選抗病毒藥物的工具，也可應用於發展疫苗。近年來，許多黃病毒的複製子系統已被建立，包括Kunjin病毒、蜱蝨傳播腦炎病毒、西尼羅病毒、黃熱病毒等。雖然登革病毒（dengue virus）的複製子也已經發展出來，但只侷限於第二型登革病毒（DENV2），且表現的效率也並不高。為了能更廣泛應用複製子，必須發展出有效且方便的組裝核殼（encapsidation）系統，將複製子組裝成似病毒顆粒（virus-like particles）。
本研究的第一個研究目標即是要建立持續產生前驅膜/外套膜蛋白質（prM/E）的次病毒顆粒的細胞株，當作組裝複製子的細胞（packaging cell lines）。第二個目標是建立第四型（DENV4）與第二型（DENV2）登革病毒的複製子。第三個目標是探討複製子組裝進入似病毒顆粒的情形。
為了表現prM/E蛋白質，建構了pCBJssD1J396、pCBJssD3J396、pCBJssD4J396三個prM/E重組質體，及用於建立持續表現prM/E蛋白質之細胞株的三個重組質體pCDJssD1J396、pCDJssD2J396及pCDJssD4J396。由免疫螢光染色可以觀察到除了pCDJssD1J396外，pCBJssD1J396、pCBJssD3J396、pCBJssD4J396與pCBJssD2J396暫時轉染至細胞以及由pCDJssD1J396、pCDJssD2J396、pCDJssD4J396建立之持續表現細胞株之E蛋白質的表現量皆很高。用西方墨點分析法也可觀察到暫時轉染及持續表現prM/E之細胞株其培養上清液經超高速離心之沈澱物（pellet lysates）中都可看到高量的prM與E蛋白質的表現，顯示所建立的prM/E重組質體及持續表現prM/E之細胞株都可有效產生prM/E次病毒顆粒。
登革病毒複製子的建立分為兩種，一種是刪除prM及大部分E基因所構築的DENV4複製子：T7C101E470NSpFast，與DENV2複製子：SP6C101E470NSp2。另一種為刪除prM與大部分E基因，以及大部C基因所構築的DENV4之複製子：T7C30E470NSpFast。將這三種複製子RNA轉染至BHK與293T細胞內以免疫螢光染色法都可偵測到NS1蛋白質的表現，顯示這三個複製子在細胞內都可以複製並表現蛋白質。而複製子在BHK細胞中的表現較有效率。T7C101E470NSpFast複製子的蛋白質表現也比T7C30E470NSpFast好。
為了探討建立好的持續表現prM/E之細胞株是否可以組裝複製子形成似病毒顆粒，將胞外轉錄之複製子 RNA轉染至pCDJssD2J396與pCDJssD2J396所建立之兩種持續表現prM/E之細胞株及暫時轉染pCDDssD2質體之293T細胞，4天後，收取培養上清液經超高速離心得到之似病毒顆粒，再感染BHK細胞，2天後再以免疫螢光染色法偵測是否有病毒NS1蛋白質的合成。結果發現只有以pCDJssD2J396所建立之持續表現細胞株為組裝系統，可以將T7C101E470NSpFast複製子組裝成具感染力的似病毒顆粒。另外兩者都不能產生有感染力之似病毒顆粒。以免疫螢光染色法觀察複製子轉染後在細胞的表現，在pCDJssD4J396所建立之持續表現細胞株或暫時轉染pCBDssD2質體的細胞內，三個複製子的表現都相當低，故可能因為RNA轉染的效率太差，使得無法組裝成似病毒顆粒。因此，究竟怎樣的複製子組裝系統是較理想的需要進一步的研究。但由實驗的結果可以知道由pCDJssD2J396所建立的持續表現prM/E之細胞株可以當作組裝DENV 複製子組裝細胞，可以成功的產生有感染力的似病毒顆粒。
本研究的整體目標即是建立出有效且方便的登革病毒複製子組裝系統，這個系統不但可應用於未來深入探討登革病毒複製過程中組裝核殼步驟的機制；由此產生之具感染力的似病毒顆粒更可以用於未來探討病毒進入細胞步驟的機制以及篩選抑制此步驟之藥物。

Subgenomic replicons of positive-stranded RNA viruses have been shown to be valuable tools for studying viral replication independently of virion assembly, screening inhibitors, expressing heterologous genes, and developing new immunization vectors. Recently, replicons of several flaviviruses have been successfully constructed, including Kunjin viruses, tick-borne encephalitis viruses, West Nile viruses, yellow fever viruses, and dengue viruses（DENV）. Although replicon of dengue virus type 2（DENV2）was reported recently, an efficient encapsidation system for dengue replicon has not been developed yet. The first specific aim of this study is to establish stable clones that produce subviral particles containing the precursor membrane and envelope （prM/E）proteins of DENV. The second aim is to establish replicons of dengue virus type 4（DENV4）and DENV2. The third aim is to study the encapsidation of replicon into virus-like particles （VLPs）by using appropriate packaging cell line.
Stable cell lines constitutively expressing prM and E proteins of DENV1, DENV2 and DENV4 were successfully established to produce subviral particles efficiently. Immunofluorescence analysis revealed high levels of expression of E protein except the DENV1 prM/E stable clone. In the pellets derived from ultracentrifugation of supernatants, expression of prM and E proteins was shown by Western blotting analysis, indicating that these prM/E stable clones were capable of producing subviral particles.
Both replicons of DENV4 and DENV2 were constructed. One type of replicons, designated as T7C101E470NSpFast（DENV4）and SP6C101E470NSp2（DENV2）, lacks the entire prM and the majority of E genes. Another type of replicon designated as T7C30E470NSpFast（DENV4）, lacks the entire prM and the majority of C and E genes. Immunofluorescence analysis revealed that these three replicons were able to replicate and express proteins in BHK cells and 293T cells, though the efficiency is not high especially in 293T cells.
To investigate whether prM/E stable clones can package DENV replicons, the in vitro-transcribed replicon RNA were transfected to prM/E stable clones（ pCDJssD2J396 and pCDJssD4J396 ）, as well as to and 293T cells transiently transfected with a construct that express prM/E of DENV2（pCBDssD2）. Four days after RNA transfection , VLPs derived from culture supernatants were inoculated to fresh BHK cells to examine the expression of NS1 protein by immunofluorescence. Only the DENV4 replicon, T7C101E470NSpFast, can produce infectious VLPs after its transfection to prM/E stable clone pCDJssD2J396. No infectious VLPs was produced in the pCDJssD4J396 stable clones and cells transfected with pCBDssD2. This could be due to low efficiency of transfection of RNA replicon, since expression of NS1 protein by T7C101E470NSpFast replicon in pCDJssD2J396 stable clone was better than that in other two prM/E expressing cells. Take together, these results indicated that dengue prM/E stable clone, pCDJssD2J396, can be used as a packaging cell line for DENV replicon to generate infectious VLPs for future study. However, the ability to package replicon for other prM/E expressing cells needs to be further studied.
The overall objective of this study is to establish an efficient and convenient packaging system for DENV replicon. The packaging system can be used to study the mechanism of the encapsidation step of DENV replication. The infectious VLPs derived from the system can be used to study the mechanism of DENV entry and to screen entry inhibitors as well.

中文摘要 1
英文摘要 3
序論 5
材料與方法 16
一、質體的構築 16
二、菌種 22
三、質體轉形作用 23
四、製備小量質體 23
五、製備大量質體 24
六、細胞培養 24
七、DNA 轉染作用 26
八、胞外轉錄 RNA 27
九、RNA 轉染作用 28
十、間接免疫螢光染色 28
十一、建立持續表現登革病毒pr M蛋白質和E蛋白質的細胞株 29
十二、西方墨點分析法 29
十三、細胞感染登革病毒實驗 30
十四、以超高速離心法分析病毒顆粒或次病毒顆粒 31
十五、複製子RNA組裝進入病毒顆粒實驗 31
結果 32
一、登革病毒prM/E重組質體之構築及表現 32
二、建立持續表現登革病毒prM/E蛋白質之細胞株 32
三、以西方墨點法分析prM/E重組質體與持續表現登革病毒prM/E蛋白 33
質之細胞株其prM及E蛋白質的表現及形成次病毒顆粒的特性
四、建立登革病毒之複製子及其表現 34
五、組裝複製子RNA形成似病毒顆粒 37
討論 39
參考文獻 66

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