- 學位論文
利用昆蟲桿狀病毒載體系統於昆蟲及哺乳細胞表現豬環狀病毒蛋白
Expression of Porcine circovirus Type 2 Capsid Protein in Insect and Mammalian Cells Using Baculovirus Expression Vector System
摘要
學號: M10022017 論文題目: 利用昆蟲桿狀病毒載體系統於昆蟲及哺乳細胞表現豬環狀病毒蛋白 總頁數: 96 學校名稱: 國立屏東科技大學 系(所)別: 熱帶農業暨國際合作系 畢業時間及摘要別: 一百零一學年度第二學期碩士學位論文摘要 研究生: 阮崇海 指導教授: 鄭力廷博士 鍾曜吉博士 鄭達智博士 論文摘要內容: 豬環狀病毒第2型 (PCV2)離乳後多系統消耗性綜合症 (PMWS)主要病源，且為當代養豬產業新興疾病 ，常造成養豬國家經濟上重大損失。因此，在多數的養豬國家急需迫切發展一有效疫苗來控制此疾病。近年，新型疫苗之開發多以較具吸引力之昆蟲桿狀病毒表現系統為。故本研究利用秋行軍蟲 (Sf9)細胞表現昆蟲桿狀病毒攜帶 PCV2 之開放閱讀框架2 (ORF2)基因，該基因片段經轉錄轉譯後為PCV2之衣核蛋白，且被證實為一重要免疫原；隨後利用巨細胞病毒啟動子轉錄帶有 ORF2基因之重組桿狀病毒，並於中國倉鼠卵巢 (CHO)細胞中表現病毒衣殼蛋白。將成功轉染至Sf9細胞及CHO細胞之重組桿狀病毒，觀察其細胞病變及綠螢光表現情形。此外，利用聚丙烯醯胺膠體 (SDS-PAGE)證實其重組蛋白表現位置與產量，以及使用西方墨點法 (Western blot)以His-tag及陽性血清 (PCV2)證實其抗原性。本研究結果顯示，已成功表現PCV2之重組衣殼蛋白於昆蟲細胞表現系統；但目前正極力將重組衣殼蛋白表現於哺乳細胞表現系統內。未來將發展一有效且新型之PCV2疫苗，得以控制PCV2之威脅。
並列摘要
Student ID: M10022017 Title of thesis: Expression of Porcine circovirus Type 2 Capsid Protein in Insect and Mammalian Cells Using Baculovirus Expression Vector System Total page: 96 Name of institute: Department of Tropical Agriculture and International Cooperation Graduate date: June 28th, 2013 Degree conferred: Master Name of student: Nguyen, Trong Hai Advisors: Li-Ting Cheng, Ph.D. Yao-Chi Chung, Ph.D. Philip Ta Cheng, Ph.D. The content of abstract in this thesis: Porcine circovirus type 2 (PCV2) has been known as the primary causative agent of post-weaning multisystemic wasting syndrome, an emerging swine disease which causes tremendous economic losses. Thus, development of a highly efficient vaccine to control this pathogen is an urgent need for the swine industry in many countries. Recently, baculovirus has emerged as an attractive gene expression system for novel vaccine technology production. In this study, recombinant baculovirus carrying open reading frame 2 gene of PCV2 first was developed to express PCV2 capsid protein, the most important immunogen of PCV2, in Spodoptera frugiperda (Sf9) cells. Additionally, the recombinant baculovirus with the presence of cytomegalovirus promoter as protein transcriptional control in mammalian cells was generated and subsequently utilized to transduce Chinese hamster ovary (CHO) cells for expression of the capsid protein. Efficiency of recombinant virus transfection and infection in Sf9 cells was investigated by inspecting the appearance of cytopathic effect on the cells and also by comparing with positive control expressing green fluorescence. The capsid protein expressed then was evaluated by SDS-PAGE and Western blot using both antibody against His-tag and PCV2. Result shows that baculovirus expression vector system (BEVS) can be employed to successfully express the PCV2 capsid protein in insect cells system. Current efforts are focused on the expression of the capsid protein in a mammalian cells system. Continuing study is needed for developing an effective novel vaccine for PCV2 infection management.
參考文獻
延伸閱讀
- 韓暉（2011）。以桿狀病毒表現載體於昆蟲細胞表現人類巨細胞病毒gB AD1及AD2蛋白質片段及其在抗體檢測上之應用〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2011.00968
- 黃子軒（2015）。分析桿狀病毒及鱗翅目昆蟲細胞之密碼子使用模式以提升昆蟲表現系統之外源蛋白表現量〔碩士論文，中原大學〕。華藝線上圖書館。https://doi.org/10.6840/cycu201500906
- 蔡立宸（2022）。以桿狀病毒表現系統表現蜜蜂囊狀幼蟲病毒之結構蛋白〔碩士論文，中原大學〕。華藝線上圖書館。https://doi.org/10.6840/cycu202200140
- 江迎瑋（2006）。以桿狀病毒/哺乳動物細胞表現系統生產D型肝炎病毒蛋白質與類病毒顆粒之研究〔碩士論文，國立清華大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0016-1303200709264447
- 涂邦彥（2014）。Expression profiles of Toll-like receptor mRNA in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2〔碩士論文，國立屏東科技大學〕。華藝線上圖書館。https://doi.org/10.6346/NPUST.2014.00161