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研究生:王嘉裕
研究生(外文):Chia-Yu Wang
論文名稱:犬瘟熱病毒血球凝集素蛋白選殖表現與單株抗體製備
論文名稱(外文):Cloning and Expression of Hemagglutinin Protein from Canine Distemper Virus and Production of the Monoclonal Antibodies
指導教授:廖明輝廖明輝引用關係
指導教授(外文):Ming-Hui Liao
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:犬瘟熱H蛋白酵素連結免疫分析單株抗體
外文關鍵詞:Canine distemper virusH proteinELISAMonoclonal antibody
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犬瘟熱病毒(Canine Distemper Virus, CDV)為造成犬隻罹患犬瘟熱之主要致病原,具高度接觸傳染性,高度發病率與高死亡率。犬瘟熱所引起的病徵包括,發燒、鼻炎、肺炎、結膜炎、腸胃炎,造成的致死率依照物種差異而有所不同。分類上犬瘟熱病毒屬於副黏液病毒科(family Paramyxoviridae),麻疹病毒屬(genus Morbillivirus),為具封套,線狀負向單股的RNA病毒。CDV基因組成主要為六個蛋白基因,分別為N、P、M、L、F、H。本研究由胺基酸序列分析比對發現,病毒的H蛋白具有相當高的抗原性,因此本實驗設計數個引子對,以RT-PCR增幅出血球凝集素蛋白(Hemagglutinin protein, H)片段,經定序後與NCBI資料庫中的序列進行比對。再將此基因構築至pET32a表現戴體,並利用原核表達系統表現重組H蛋白,再將此重組H蛋白與全病毒分別免疫BALB/c小鼠,取其血清進行免疫呈色分析,由結果來證實本研究所表現的重組H蛋白不但正確,也具有抗原性。隨後將免疫的BALB/c鼷鼠之脾細胞與骨髓瘤細胞(NS-1)進行融合來產生融合瘤,來製備H蛋白的單株抗體,經ELISA、Western-blot、Immuno-dot分析確認後,共篩得7株抗體,所篩得的皆是對抗直線形抗原決定位之抗體,其中一株為對抗pET32a之Tag的抗體,其餘6株為對抗H蛋白之抗體,此6株可辨別到病毒態H蛋白,未來抗體可應用於ELISA檢測套組的開發上。
(Canine Distemper Virus, CDV) cause dogs to suffer from dog's acute communicable diseases. it is infective to highly keep in touch, high incidence of disease and high death rate and causing the etiology mainly. CDV is a member of the genus Morbillivirus of the family Paramixoviridae. CDV is an enveloped virus with single stranded, linear, non-segmented, and negative-sense RNA, it contained six genes in order 3’-N-P-M-F-H-L-5’. In this study, H protein has higher antigenicity in comparison with amino acid sequence of other CDV. The H gene fragment of CDV was amplified using reverse transcription polymerase chain reaction (RT-PCR) and PCR products were confirmed by DNA sequencing. Subsequently, The H gene was subcloned into prokaryotic expression vector (pET32a) and transformed into E.coli (BL-21 (DE3) pLysS) for expression. The expressed protein was analyzed by SDS-PAGE and purified by affinity column then immunize BALB/c mice. The anti-serum of purified H protein was needed to recognize the H protein of CDV by Western blot. The myeloma cells (NS-1) will fuse with spleen cells from immune BALB/c mice to produce hybridoma cells. The expressed H protein will be coated as antigen. Antibodies secreted from hybridoma cells were screened by ELISA, Western-blot, and Immuno-dot. There are 7 monoclonal antibodies prepared in this study, including 1 anti-pET32a-Tag and 6 anti-H protein antibodies. Immuno-dot and Western-blot analyses suggested that these 7 monoclonal antibodies recognized a linear epitope. The 6 monoclonal antibodies which anti expressed H protein can recognize the H protein of virus. These monoclonal antibodies can be used in further research to develop CDV testing reagent.
目 錄
第1章
前言.......................................................1
第2章
前人研究....................................................2
2.1 犬瘟熱的歷史背景及流行病學.............................2
2.2 犬瘟熱病毒之病毒特性..................................3
2.3 犬瘟熱病毒細胞感染過程概述.............................4
2.4 犬瘟熱病毒之致病機轉..................................4
2.5 犬瘟熱病毒之臨床症狀..................................6
2.6 犬瘟熱病毒血液凝集素蛋白之探討.........................7
2.7 酵素連結免疫吸附測定(ELISA)..........................8
2.8 單株抗體.............................................8
2.8.1 單株抗體之歷史背景.................................8
2.8.2 細胞融合的方式與篩選原理............................9
2.8.3 單株抗體之特性與應用..............................10
第3章
材料與方法.................................................15
3.1 實驗材料............................................15
3.1.1 病毒株..........................................15
3.1.2 細胞株..........................................15
3.1.3 菌種與載體.......................................15
3.2 細胞培養與病毒的增殖.................................15
3.2.1 細胞培養.........................................15
3.2.2 細胞的繼代培養...................................16
3.2.3 細胞的冷凍保存...................................16
3.2.4 病毒的增殖.......................................16
3.3 犬瘟熱病毒血液凝集素蛋白基因的選殖.....................17
3.3.1 病毒核酸的萃取................................17
3.3.2 全長H基因的選殖引子設計........................17
3.3.3 反轉錄聚合酶鏈鎖反應(RT-PCR)...................17
3.3.4 DNA電泳分析...................................18
3.3.5 PCR產物之純化.................................18
3.3.6 接合作用(Ligation)............................18
3.3.7 勝任細胞(competent cell)之製備................19
3.3.8 轉形作用(Transformation)......................19
3.3.9 菌落之篩選....................................19
3.3.10 小量質體DNA之萃取與純化.......................20
3.3.11 限制酶切割確認重組之質體DNA....................20
3.3.12 菌種的保存...................................20
3.3.13 核酸之定序與分析..............................21
3.4 犬瘟熱病毒血液凝集素蛋白的表現......................21
3.4.1 引子設計......................................21
3.4.2 反轉錄聚合酶鏈鎖反應(RT-PCR)...................22
3.4.3 PCR產物之純化.................................22
3.4.4 接合作用(Ligation)............................23
3.4.5 轉形作用與菌落之篩選...........................23
3.4.6 小量質體DNA之萃取與純化........................23
3.4.7 限制酶切割確認重組之質體DNA.....................23
3.4.8 表現載體的選擇及處理...........................23
3.4.9 接合作用(Ligation)............................24
3.4.10 轉形作用(Transformation).....................24
3.4.11 菌落之篩選...................................24
3.4.12 小量質體DNA之萃取與純化.......................24
3.4.13 限制酶切割確認重組完成之質體DNA..................24
3.4.14 蛋白質的表現.................................24
3.4.15 菌體蛋白質的抽取..............................25
3.4.16 SDS-PAGE蛋白質電泳...........................25
3.5 蛋白質之純化.....................................26
3.5.1 pET32a表現的重組蛋白之純化.....................26
3.6 蛋白質的濃縮與透析................................26
3.7 蛋白質定量.......................................27
3.8 多株抗體之製備...................................27
3.8.1 犬瘟熱病毒血液凝集素蛋白抗血清之製備.............27
3.8.2 犬瘟熱病毒的陽性血清之製備......................27
3.9 蛋白質轉漬分析法(Western-blot assay).................28
3.10 單株抗體的製備......................................29
3.10.1 骨髓瘤細胞的培養.................................29
3.10.2 細胞融合........................................29
3.10.3 融合瘤細胞的培養.................................30
3.10.4 融合瘤細胞的篩選.................................30
3.10.5 融合瘤細胞的單株化...............................30
3.10.6 融合瘤細胞的再篩選...............................31
3.10.7 抗體之分析......................................31
3.10.7.1 蛋白質轉漬分析法(Western-blot assay).......31
3.10.7.2 免疫墨點法(Immuno-dot assay)..............31
3.10.7.3 抗體與病毒H蛋白之反應......................31
3.10.7.4 抗體的亞型分析(Isotyping).................32
3.10.8 抗體之生產與純化.................................32
3.10.8.1 腹水生產法................................32
3.10.8.2 抗體的純化................................33
第4章
結果......................................................34
4.1 犬瘟熱病毒H基因的分析與比對.........................34
4.1.1 全長H基因的選殖與定序分析.......................34
4.1.2 核酸序列與胺基酸序列的比對......................34
4.1.3 核酸序列與胺基酸序列的相似相異度之分析...........34
4.1.4 抗原性的分析與比較.............................36
4.2 犬瘟熱病毒H蛋白的選殖與表現.........................39
4.2.1 H蛋白的全長及片段基因之選殖與表現載體之構築.......39
4.2.2 重組蛋白的表現................................41
4.2.3 收集不同誘導時間的2個重組表現蛋白................43
4.3 重組蛋白的純化與定量..............................43
4.4 重組蛋白抗原性的證明..............................47
4.5 融合瘤細胞之製備...................................50
4.6 融合瘤細胞之篩選與單株化...........................51
4.7 單株化之後的抗體篩選..............................52
4.8 抗體亞型分析........................................56
4.9 抗體之生產與純化.....................................57
第5章
討論......................................................58
參考文獻...................................................62
附錄......................................................69
作者簡介...................................................75
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