臺灣博碩士論文加值系統

非洲豬瘟 (African swine fever；ASF) 是由非洲豬瘟病毒 (African swine fever virus；ASFV) 引起的惡性動物傳染病。自1921年首次在非洲肯亞報導以來，已傳播至三大洲，近年歐洲及亞洲各國陸續爆發疫情，造成全球養豬產業重大損失。至今仍無有效的疫苗及治療方法。ASFV為雙股DNA病毒，基因長度約為170至193 kbp，包含151至167個開放閱讀框，編碼超過150種蛋白，多數蛋白的功能未知。p72蛋白的編碼基因為B646L，是組成ASFV衣殼的主要結構蛋白，在病毒感染的後期表達，也是受感染豬隻被檢測到的主要抗原之一。p72蛋白所誘發的單株抗體已被證實有中和病毒的能力，並阻止ASFV與巨噬細胞結合。因此被認為可用來作為疫苗開發及血清學診斷之抗原。pB602L蛋白的編碼基因為B602L，為ASFV晚期表現之非結構蛋白，目前所知的功能為幫助p72蛋白完整折疊，形成正確的三聚體結構，是謂p72蛋白的伴護蛋白 (chaperone protein)。本論文研究目標為利用昆蟲桿狀病毒表現系統進行ASFV 結構蛋白p72與非結構蛋白pB602L之表達，以提供基礎科學研究與診斷試劑研發之需求。本實驗將B646L基因選殖至pENTR™/D-TOPO載體中，製備帶有His tag基因的Topo-p72-His質體，藉由Gateway Technology進行桿狀病毒基因重組。同時將帶有His tag基因的B646L基因和帶有Flag tag基因的B602L基因選殖至pOET5載體中，製備pOET5-co-express質體，並透過同源重組方式將目標基因轉移到桿狀病毒基因組中。利用西方墨點和免疫螢光染色法來分析重組蛋白。結果顯示p72和pB602L重組蛋白在桿狀病毒系統中成功共同表達。這些結果將有助於進一步研究與開發診斷試劑。

African swine fever (ASF) is a malignant animal infectious disease caused by African swine fever virus (ASFV). In recent years, there have been outbreaks of epidemics in various countries, causing significant losses to the global pig industry. Currently, there is still no effective vaccine and treatment for ASF. ASFV is a large, double stranded DNA virus that causes a highly lethal disease in domestic pigs, and its genome ranges in length from about 170 to 193 kbp that encodes 151 to 167 ORFs. p72 is the major structure protein encoded by the gene B646L of ASFV and is very important for forming the viral capsid in the late stage of viral infection. p72 also is one of the major antigens detected in infected pigs. Monoclonal antibodies recognizing p72 were shown to neutralize virulent ASFV isolates and to prevent ASFV binding to macrophages. pB602L is a non-structural protein expressed in the late stage of ASFV infecton. It serves as a chaperone for the folding of p72 to form the trimmer structure and prevents the aggregation of p72. The aim of this study is to co-express p72 and pB602L proteins by baculovirus system and to develop diagnostic techniques as ASFV control strategies in Taiwan. Two baculovirus expression vectors, Topo-p72-His and pOET5-co-express, were constructed in the present study. The gene B646L was cloned into pENTR™/D-TOPO vector which contained a hexahistidine tag sequence to generate Topo-p72-His plasmid and transferred into the baculovirus genome by the Gateway Technology. The gene B646L with a hexahistidine tag sequence and the gene B602L with flag tag sequence were cloned into pOET5 vector to generate pOET5-co-express plasmid and transferred into the baculovirus genome by homologous recombination. Western blotting and immunofluorescence assay were applied to analyze recombinant proteins. The results showed that p72 and pB602L were successfully co-expressed by the baculovirus system. These results will aid in further research for development diagnostic techniques.

摘要 i
Abstractii
目次 iii
表目次 vi
圖目次 vii
第一章 前言 1
第二章 文獻回顧 2
第一節 非洲豬瘟病毒 2
2.1.1 歷史簡介 2
2.1.2 病毒構造 3
2.1.2.1 p72蛋白 3
2.1.2.2 pB602L蛋白 4
2.1.3 病毒複製 5
2.1.4 傳播方式 5
2.1.5 實驗室診斷方法 7
2.1.5.1 抗原檢測 7
2.1.5.2 抗體檢測 8
2.1.5.3 側向流動試驗法 (lateral flow assay) 9
第二節 昆蟲桿狀病毒表現系統 9
2.2.1 桿狀病毒簡介 9
2.2.2 桿狀病毒生活史 10
2.2.3 桿狀病毒基因表現 10
2.2.4 桿狀病毒表現系統發展與應用 11
2.2.5 flashBACTM Baculovirus Expression System 11
2.2.6 BaculoDirect™ Baculovirus Expression System 12
2.2.7 Bac-to-Bac™ Baculovirus Expression System 12
2.2.8 研究動機與目的 13
第三章 材料與方法 14
第一節 實驗目的 14
第二節 表現p72重組蛋白 14
3.2.1 核酸引子設計 14
3.2.2 聚合酶鏈鎖反應 15
3.2.3 瓊脂糖膠體電泳 15
3.2.4 膠體純化 15
3.2.5 質體構築 16
3.2.6 轉型作用 16
3.2.7 重組Topo-p72-His質體之篩選 16
3.2.8 序列分析 16
3.2.9 菌種保存 16
3.2.10 質體萃取 17
3.2.11 桿狀病毒基因重組 17
3.2.12 昆蟲細胞之轉染作用 17
第三節 共同表現p72與pB602L重組蛋白 18
3.3.1 核酸引子設計 18
3.3.2 聚合酶鏈鎖反應 18
3.3.3 限制酶切割反應 19
3.3.4 接合反應 19
3.3.5 轉型作用 19
3.3.6 重組pOET5-co-express質體之篩選 19
3.3.7 昆蟲細胞之轉染作用20
第四節 昆蟲細胞培養 20
3.4.1 細胞培養 20
3.4.2 細胞冷凍 21
3.4.3 細胞解凍 21
第五節 確認重組病毒帶有目標基因 21
第六節 重組病毒純化及力價測定 22
第七節 蛋白質電泳 22
第八節 蛋白質電泳膠片染色 22
第九節 西方墨點法 23
第十節 免疫螢光染色 23
第四章 結果 24
第一節 重組蛋白p72及pB602L之基因選殖 24
第二節 重組質體之定序結果 24
第三節 重組桿狀病毒DNA轉染Sf21細胞 24
第四節 重組桿狀病毒帶有目標基因之檢測 24
第五節 p72及pB602L重組蛋白西方墨點法之分析 25
第六節 p72及pB602L重組蛋白免疫螢光染色法之分析 25
第五章 討論 26
第六章 參考文獻 30
附錄一、Gateway 重組反應 46
附錄二、pENTR™/D-TOPO®圖譜 47
附錄三、BaculoDirectTM C-Term Linear DNA圖譜 48
附錄四、第一型拓樸異構酶在pENTR™/D-TOPO®的作用機制 49
附錄五、pOET5圖譜 50
附錄六、Topo-p72-His與pOET5-co-express質體之p72胺基酸序列比對結果 51
附錄七、pOET5-co-express 質體之pB602L胺基酸序列比對結果 52

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