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研究生:賴以斌
研究生(外文):Yi-Pin Lai
論文名稱:以噬菌體展示技術搜尋能與鼻咽癌細胞株具有結合力之分子
論文名稱(外文):Searching for Molecules Capable of Binding to Nasopharyngeal Carcinoma Cell Lines Using Phage Display Technology
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:63
中文關鍵詞:噬菌體展示
外文關鍵詞:Phage Display
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噬菌體展示系統是將外來蛋白表現於以外殼蛋白為骨架的噬菌體表面。當將一群序列不同的外來基因插在外殼蛋白基因的5’端,則可製備出噬菌體基因庫,在大腸桿菌中表現與組裝後,得到的重組噬菌體能表現外來蛋白且其基因就在此噬菌體上,藉由親和選擇的過程,與標的物具有結合力之分子可由噬菌體展示庫中篩選出來。本研究的目的即在於以噬菌體展示庫篩選出能與鼻咽癌細胞株具有結合力之抗體或胜鈦分子。
為了得到鼻咽癌細胞專一的抗體,以鼻咽癌細胞NPC-076免疫小鼠,將得到的VH及VL抗體基因片段轉殖到phh3-stuffer與J558VL stuff phh3muγ1載體,分別得到大小為4.8×105及1.68×106的phh3-Fab與phh3-Fab(λ)噬菌體展示庫,但並未由此二展示庫篩選到鼻咽癌細胞專一的抗體。為了得到NPC-076細胞專一的胜鈦,以Ph.D.-C7C逢機胜鈦庫依序對NPC-076進行正向淘選及對肝癌細胞株進行負向淘選,親和選擇後,將個別的噬菌體增殖並以ELISA測定其對NPC-076的結合力。在一親和選擇策略中,在總數422個單一溶菌斑中逢機挑選100個進行篩選,得到8株對鼻咽癌細胞株NPC-076具有顯著性結合的選殖株。除NPC-076之外,這8株重組噬菌體對乳癌細胞株BT-20、肝癌細胞株Huh-7也具有結合力。這8個選殖株中,以編號164的噬菌體對各測試的癌細胞具有最強的結合力。序列分析結果顯示編號150及164的噬菌體是一樣的。雖然這7個選殖株沒有保守序列,但幾乎每個選殖株的逢機胜鈦序列區域都出現一或二個serine胺基酸,推測其氫氧基團在胜鈦與細胞膜結合上扮演重要角色。
In phage display system, foreign proteins are displayed on the surface of phage particles using one of the coat proteins as the scaffold. A library is obtained by inserting a batch of DNA fragments with different sequences into the 5’-end of the coding region of the coat protein. Upon expression and subsequently assembly, each phage particle displays a distinct foreign target on the phage surface with its genetic material residing within the phage particle. In conjunction with affinity selection, molecules with desired binding property can be readily obtained from phage display libraries. The aim of this study is to search for molecules, antibodies or peptides, capable of binding to nasopharyngeal carcinoma (NPC) cell lines to be used as the targeting agents.
To generate NPC cell specific antibodies, two phage display libraries, phh3-Fab and phh3-Fab(λ) with 4.8×105 and 1.68×106 members, respectively, were generated from NPC-076 cell-immunized BALB/c mice. No phage-Fab specific for NPC-076 cell line was isolated from these two libraries. To obtain peptides specific for NPC-076 cell lines, phage display random peptide libraries Ph.D.-C7C was subjected to negative and positive selections with hepatocellular carcinoma (HCC) cell lines and NPC-076, respectively. After affinity selection, individual phage was propagated separated and its binding to NPC-076 was evaluated by ELISA. In one of the affinity selection strategies, eight individual phages with significant binding to NPC-076 were isolated out of 100 randomly selected clones from total 422 output plaques. In addition to NPC cell lines, these eight recombinant phages also bound to a breast cancer cell line BT-20 and a hepatocellular carcinoma cell line Huh-7. Among these 8 clones, clone #164 showed the highest binding capacity to all cell lines tested. Sequence analysis revealed that phages #164 and #150 are in fact the same. Although no consensus sequence can be depicted from these seven sequences, serine or threonine residue was found in all peptides suggesting that hydroxide group may play an important role in the interactions between the peptide and cell membrane.
縮寫字對照表……………………………………………………………………...1
中文摘要…………………………………………………………………………...3
英文摘要…………………………………………………………………………...4
壹、前言……………………………………………………………………………5
I.鼻咽癌之成因、診斷與治療………………………………………………….5
II.噬菌體展示技術……………………………………………………………...6
貳、材料與方法…………………………………………………………………….11
I.實驗材料……………………………………………………………………….11
一、菌株與細胞株……………………………………………………………11
二、培養基與溶液配方………………………………………………………11
三、引子………………………………………………………………………16
II.實驗方法……………………………………………………………………...16
一、質體DNA之製備………...……………………………………………..16
二、質體DNA之切割……………………………………………………….17
三、DNA洋菜膠體電泳分析……..…………………………………………17
四、DNA片段之回收與純化………..………………………………………17
五、DNA黏接反應…………………………………………….…………….17
六、勝任細胞之製備………..………………………………………………18
七、質體轉形…………………...……………………………………………18
八、癌細胞之培養 ………………………………………………………….19
九、小鼠之免疫試驗...………………………………………………………19
十、酵素連結免疫呈色法…………...……….……………………………..20
十一、小鼠組織、器官Total RNA之製備….……………………………...20
十二、反轉錄反應…………….……………………………………………..21
十三、聚合脢鏈反應…………………………………………………………21
十四、重組噬菌體及輔助噬菌體之製備、沈澱與計數….………………….22
十五、親和選擇……………………………………………………………...23
十六、Ph.D.噬菌體逢機胜鈦庫之親和選擇及重組噬菌體之製備………..23
十七、DNA定序分析……………………………………………………….24
十八、蛋白質之分析…………………………………………………………25
參、結果與討論……………………………………………………………………28
I. 噬菌抗體基因庫之構築與親和選擇…………………………………...……28
一、小鼠之免疫試驗……………………………………………………….28
二、phh3-Fab抗體基因庫之構築與篩選………………………………….28
三、phh3-Fab(λ)噬菌抗體基因庫之構築與篩選………………………….30
II.搜尋癌細胞結合胜鈦………………………………………………………...31
一、以癌細胞對Ph.D.-C7C逢機胜鈦庫進行親和選擇……………………31
二、Ph.D.-C7C噬菌胜鈦選殖株對癌細胞結合力之分析…………………31
三、Ph.D.-C7C噬菌胜鈦選殖株之序列分析………………………………32
四、Ph.D.-C7C噬菌胜鈦選殖株之西方轉漬法分析………………………33
五、以癌細胞對混合之逢機胜鈦庫進行親和選擇………………………….33
III.結論…………………………………………………………………………..34
肆、參考文獻…………………….………………………………………………...36
圖表……………………………………………………………...…………………42
表一、本實驗所使用之菌株。………………………………………………...42
表二、聚合脢鏈反應所使用的引子。………………………………………...43
圖一、以ELISA測試三隻BALB/c小鼠中血清抗體效價與免疫時間關係圖。…………...………………………………………………………….
45
圖二、VL-VH基因合成流程圖。……………………………………………..46
圖三、抗體變異區基因first PCR產物之洋菜膠體電泳分析。……………...47
圖四、小鼠phh3-VL-plppl-VH抗體基因庫之構築。………………………..48
圖五、改良後之VL與VH基因合成流程圖。………………………………49
圖六、phh3-Fab抗體基因庫之構築。………………...………………………50
圖七、以DNA洋菜膠體電泳分析逢機挑選之phh3-VH與phh3-Fab抗體基因庫選殖株。…………………………………………………………
51
圖八、phh3-Fab(λ)抗體基因庫之構築。……………………………...………52
圖九、以DNA洋菜膠體電泳分析逢機挑選之phh3-Fab(λ)抗體基因庫選殖株。……………………………………………………………………
53
圖十、以NPC-076對phh3-Fab(λ)噬菌抗體庫進行五次親和選擇後所得到的重組噬菌體數目。…………………………………………………...
54
圖十一、phh3-Fab(λ) soluble Fab抗體之表現載體構築。….………………...55
表三、以不同癌細胞對不同噬菌胜鈦庫進行不同親和選擇策略之結果。…56
圖十二、Ph.D.-C7C噬菌胜鈦選殖株對NPC-076的結合能力。……...……57
圖十三、Ph.D.-C7C噬菌胜鈦選殖株對不同癌細胞之結合能力。………….58
圖十四、Ph.D.-C7C噬菌胜鈦選殖株對癌細胞之劑量依賴效應。………….59
表四、Ph.D.-C7C噬菌胜鈦選殖株之核酸及胺基酸序列。………….……..60
表五、以BLAST軟體分析Ph.D.-C7C噬菌胜汰選殖株之逢機胜鈦區域。..61
圖十五、12.5% SDS-PAGE及西方轉漬法分析。……………………………62
表六、以癌細胞對混合之Ph.D.-C7C、-7、-12逢機胜鈦庫進行親和選擇後所得到的噬菌體數目。………………………………………………
63
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