臺灣博碩士論文加值系統

台灣每年約有500 ~ 1000人被毒蛇咬傷，注射抗蛇毒血清是目前用以治療被蛇毒咬傷最有效的方法。目前抗蛇毒血清的製備主要以去毒性蛇毒免疫馬匹後精製而得。然而，在以馬匹製造抗蛇毒血清過程中，需歷經3到5個月的去毒性蛇毒針劑注射，常導致馬匹逐漸消瘦甚至死亡，因此研發新式有效的蛇毒抗體是一項重要課題。本實驗主要利用噬菌體呈現蛇毒擬抗原決定位免疫小鼠產生抗體以及基因工程產生單鏈變異區片段抗體(single-chain variable fragment, scFv)兩種方式，開發新的製造抗眼鏡蛇蛇毒抗體的方法。在噬菌體呈現蛇毒擬抗原決定位方面，將先前篩選出八個代表眼鏡蛇蛇毒不同抗原決定位的擬抗原噬菌體免疫小鼠後的抗血清混合，進行中和眼鏡蛇蛇毒能力的試驗，結果發現小鼠存活率為100％，中和效力為16 TU (Tanaka unit)。在單鏈變異區片段抗體製備方面，從具有抗眼鏡蛇蛇毒抗體的小鼠脾臟細胞萃取mRNA，以聚合酶鏈連鎖反應擴增出抗體重鏈及輕鏈的變異區基因，建構出單鏈變異區片段抗體基因，將此抗體基因接合到pET101載體後轉形至大腸桿菌中大量表現抗體蛋白。將這些抗體蛋白混合後，利用動物實驗分析中和蛇毒效果，結果顯示小鼠存活率為50％，中和效力為8TU。綜合上述結果顯示，此兩種方式生產之抗體具有中和蛇毒蛋白能力，利用這兩種策略製造抗體，可以改進傳統製造抗蛇毒血清需耗費大量時間、金錢、人力等缺點，並提供製造抗蛇毒血清可行的替代路徑。

It is about 500 ~1000 people bitten by venomous snake in Taiwan every year. The most effective method used to treat the wound by venomous snake is the injection of antivenin. So far, the preparation of antivenin is by injecting horse with detoxified snake venom and then purifies the antiserum. However, it needs to take 3~5 months to immunize the horse for the production of antiserum. It usually causes horse being emaciated gradually and even leads to die. Therefore, it is a crucial topic to develop a novel effective anti-snake venom antibody. Two strategies were used in this study for this purpose. One is the production of antisera using phages displaying the snake venom mimotope on their coat protein as antigen, the other is the production of single chain variable fragment (scFv) antibody by gene engineering. Previously isolated eight phage clones which carrying various mimotopes of Taiwan cobra snake venom were selected to immunize mice to produce antisera. The pooled antisera were used for the cobra snake venom neutralization test. The results showed that 100% survival of the testing mice and equal to 16 TU (Tanaka unit) of neutralizing efficacy. For scFv antibody preparation, mRNA was isolated from the splenocytes of mouse which had high antibody titer against cobra snake venom. The genes encode for variable regions of heavy chain and light chain of antibodies were amplified by PCR to construct the scFv antibody genes. The scFv gene was ligated to pET101 vector and transformed to E. coli for the overexpression of scFv antibody protein. The pooled scFv antibodies were used to analyze the neutralizing efficacy against cobra snake venom in vivo. The results showed that 50% survival of the testing mice and equal to 8 TU (Tanaka unit) of neutralizing efficacy. To sum up, the antibodies produced by above two strategies have the neutralizing capability against snake venom. These strategies could improve the disadvantages including time-consuming, expensive, and labor- intensive of traditional antivenin production and provide alternative pathway for the production of antivenin.

目錄.....................................................................................................Ⅰ
表目錄......................................................................................................Ⅴ
圖目錄......................................................................................................Ⅵ
附錄目錄..................................................................................................Ⅶ
中文摘要....................................................................................................1
英文摘要....................................................................................................2
第一章 前言............................................................................................3
1.1蛇毒簡介..............................................................................................3
1.1.1眼鏡蛇(Naja naja atra).....................................................................4
1.2眼鏡蛇蛇毒...........................................................................................5
1.2.1心臟毒素(Cardiotoxin,CTX)…….....................................................5
1.2.1.1心臟毒素的分類.............................................................................7
1.2.2神經毒素............................................................................................7
1.2.2.1神經毒素分類……………….........................................................8
1.2.3磷脂質水解酵素A2...........................................................................9
1.2.3.1磷脂質水解酵素A2分類..............................................................10
1.2.4蛇毒蛋白水解酵素..........................................................................11
1.2.4.1絲胺酸蛋白水解酵素...................................................................11
1.2.4.2金屬蛋白水解酵素.......................................................................12
1.3蛇毒咬傷處置.....................................................................................14
1.4抗蛇毒血清製備與效價檢測.............................................................15
1.5噬菌體.................................................................................................16
1.5.1 M13噬菌體..................................................................................17
1.6噬菌體呈現技術.................................................................................18
1.6.1噬菌體呈現系統型式......................................................................19
1.6.2噬菌體呈現胜肽庫與抗原決定位..................................................20
1.6.3噬菌體呈現技術在蛇毒蛋白上的應用..........................................21
1.7免疫球蛋白(immunoglobulin) ..........................................................22
1.7.1免疫球蛋白的結構與功能簡介......................................................22
1.7.2重組抗體(Recombinant antibody) ..................................................24
1.7.3連接子(Linker) ...............................................................................25
1.7.4重組抗體之應用..............................................................................26
1.8研究目的.............................................................................................27
第二章 材料與方法............................................................................28
2.1材料.....................................................................................................28
2.1.1培養基及培養液之配方..................................................................28
2.1.2緩衝液及實驗試劑之配置..............................................................29
2.2方法.....................................................................................................32
2.2.1眼鏡蛇蛇毒之無毒化......................................................................32
2.2.2動物免疫..........................................................................................32
2.2.2.1去毒性眼鏡蛇蛇毒免疫小鼠.......................................................32
2.2.2.2噬菌體免疫小鼠...........................................................................33
2.2.3抗血清製備......................................................................................33
2.2.4酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA) ....................................................................................................34
2.2.5免疫後小鼠脾臟細胞之取得..........................................................34
2.2.6小鼠脾臟細胞mRNA抽取.............................................................35
2.2.7 cDNA合成......................................................................................36
2.2.8聚合酶連鎖反應(polymerase chain reaction, PCR) ......................36
2.2.8.1抗體重鏈及輕鏈變異區基因PCR選殖......................................36
2.2.8.2抗體重鏈及輕鏈變異區基因組裝...............................................37
2.2.8.3完整單鏈抗體基因PCR放大......................................................37
2.2.9洋菜膠電泳(agarose gel electrophoresis) .......................................38
2.2.10目標蛋白DNA之純化..................................................................38
2.2.11單鏈抗體基因與質體接合反應....................................................39
2.2.12質體轉型(transformation) ............................................................39
2.2.13 Colony PCR進行質體DNA選殖.................................................40
2.2.14質體DNA之純化..........................................................................40
2.2.15目標蛋白表現................................................................................41
2.2.16融合蛋白之純化............................................................................41
2.2.17 SDS-PAGE分析............................................................................42
2.2.18西方點墨法分析(Western blot analysis) ......................................43
2.2.19噬菌體之放大培養........................................................................43
2.2.20噬菌體效價測定............................................................................44
2.2.21以西方點墨法測試抗體與蛇毒接合能力(Binding assay)..........44
2.2.22動物中和測定法............................................................................45
2.2.22.1蛇毒抗原之毒力測定(Minimal Lethal Dose) ...........................45
2.2.22.2抗蛇毒血清抗體效價測定.........................................................46
第三章 結果..........................................................................................47
3.1蛇毒免疫小鼠抗體效價.....................................................................47
3.2抗體重鏈(Heavy chain)及輕鏈(Light chain)基因放大及scFv DNA
重組....................................................................................................47
3.3單鏈抗體基因質體之建構與確認.....................................................48
3.4單鏈抗體基因在原核細胞之表現與純化.........................................48
3.5單鏈抗體胺基酸分析.........................................................................49
3.6以ELISA分析擬抗原噬菌體免疫小鼠後血清抗體與單鏈抗體....50
3.7抗體蛋白接合試驗(antibody protein binding assay) ........................50
3.8動物中和測定.....................................................................................51
3.8.1蛇毒抗原之毒力測定(Minimal Lethal Dose；MLD) .....................51
3.8.2抗蛇毒血清抗體效價測定..............................................................51
表..............................................................................................................53
圖..............................................................................................................59
第四章 討論..........................................................................................72
參考文獻..................................................................................................77
附錄..........................................................................................................87

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