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研究生:林美薇
研究生(外文):Lin, Mei-Wei
論文名稱:合成生物技術應用於基因載體設計及細胞調控
論文名稱(外文):Application of synthetic biology in vector design and cell engineering
指導教授:胡育誠胡育誠引用關係
指導教授(外文):Hu, Yu-Chen
口試委員:吳肇卿陳韻晶喻秋華江啟勳
口試委員(外文):Wu, Jaw-ChingChen, Yun-ChingYuh, Chiou-HwaChiang, Chi-Shiun
口試日期:2020-07-15
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:117
中文關鍵詞:桿狀病毒載體凋亡抗體去岩藻糖醣基化
外文關鍵詞:Baculovirusvectorapoptosisantibodydefucosylated
相關次數:
  • 被引用被引用:0
  • 點閱點閱:208
  • 評分評分:
  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:0
合成生物學是一種將基因工程應用在生物系統的方法及工具,從基因體組成、調控、訊息傳遞延伸到細胞設計甚至是整個生物系統的改造。在這個研究裡,我們將合成生物技術應用於兩類生物系統-癌症治療基因載體的設計以及抗體生產細胞的改造。在第一部份-以合成開關控制的桿狀病毒載體用以調控外源基因表達及選擇性毒殺肝癌細胞: 桿狀病毒所發展的抗癌基因載體對於肝癌的治療有相當好的潛力,但應用到生物體內時,常無法避免抗癌基因也在正常細胞表現並產生毒性,為了提高安全性,我們設計了能夠感應細胞特有miRNA圖譜的miRNA感應器,並與RNA結合蛋白-L7Ae組裝成外源基因轉譯的開關,裝載到桿狀病毒載體,研究結果顯示重組的桿狀病毒載體能有效地進入肝細胞和正常細胞,但外源基因僅會在肝癌細胞中的啟動,當使用促凋亡的hBax作為外源基因時,桿狀病毒載體能在肝癌細胞和正常細胞共培養時具選擇性殺死肝癌細胞,這些結果證明了帶有合成開關元件的桿狀病毒載體,能辨識癌細胞執行專一性毒殺。在第二部分-利用CRISPR/Cas13系統進行CHO細胞株多基因調控增加去岩藻糖抗體的產量及活性: 中國倉鼠卵巢 (CHO)細胞是最廣泛使用在生產生物藥的細胞工廠,我們利用CRISPR-Cas13d RNA靶向系統進行CHO細胞系統的調控編輯,我們證實CRISPR-Cas13d有效地抑制LDHA, GFT, DDIT3以及CLU等基因,並造成功能上的改變,包含: 乳酸的產生、岩藻糖醣基化程度、氧化自由基的含量及細胞團塊的形成。再者,結合Sleeping Beauty system使CRISPR-Cas13d模組更穩定在CHO細胞株達到多重基因同時調控並證明整體抗體產量顯著提高,且降低抗體岩藻糖醣基化促使抗體依賴性細胞毒殺效果提升。展現Cas13d在CHO細胞系細胞工程中的應用潛能。綜合上述結果,在生物系統的架構裡,透過裝載合成的外源基因組件,我們優化病毒載體的效能及安全性應用於肝癌治療,並且編輯CHO細胞轉錄體增加抗體活性及產量。
Synthetic biology is a tool of applying genetic engineering to biological systems, which comprises genome manipulation, regulation, signal transduction, cell engineering or programming of entire biological systems. In this study, we applied synthetic biotechnology to cancer therapeutic vector design and engineering of antibody-producing cells. Part I-Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells: Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells and leaky anticancer gene expression. To improve the safety, we employed a miRNA sensor and assembled it with RNA binding protein, L7Ae, for BV design. Our result showed the recombinant BV efficiently entered HCC and normal cells, enabled switching ON transgene exclusively in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and normal cells for selective killing of HCC cells. Part II-Enhancing the yield and activity of defucosylated antibody produced from CHO-K1 cells using Cas13-mediated multiplex gene targetin: Chinese hamster ovary (CHO) cells are the most widely used in biopharmaceutical cell factories. We have demonstrated CRISPR-Cas13d effectively modulate the expression of several endogenous gene such as LDHA, GFT, DDIT3 and CLU, which also gave rise to the inhibition of lactic acid production, fucosylation, the reactive oxygen species generation, and cell aggregation. Furthermore, exploitation of Sleeping Beauty system-mediated integration of CRISPR-Cas13d steadily suppressed multiplex gene expression in CHO cell and endow the cell with high productivity and trimming of antibody fucosylation relevant to the enhancement of antibody-dependent cell-mediated Cytotoxicity. Collectively, we already implanted the synthetic device into the vector or organism construction for optimizing the effectiveness of viral vectors in liver cancer treatment and engineering the CHO cell transcriptome to increase antibody yield and activity.
致謝 I
摘要 III
Abstract V
目錄 VII
圖目錄 XII
Part 1. 以合成開關控制桿狀病毒載體調控外源基因表達及選擇性毒殺肝癌細胞 1
1-1序論 2
1-2文獻回顧 4
1-2-1 桿狀病毒表現系統 4
1-2-1-1 桿狀病毒在昆蟲細胞表現系統之應用 5
1-2-1-2 桿狀病毒在哺乳動物細胞表現系統之應用 6
1-2-2 miRNA系統 8
1-2-2-1 微小RNA (microRNAs; miRNAs) 8
1-2-2-2 病毒載體應用於miRNAs的傳遞 9
1-2-2-3 以miRNAs為基礎的治療方法 10
1-2-3 結合miRNA與合成開關發展新的治療型載體 11
1-2-4肝細胞癌 (Hepatocellular Carcinoma, HCC) 12
1-2-4-1 背景 12
1-2-4-2 微小RNA (miRNA)在肝臟疾病的作用 13
1-2-4-3 miR-196在生理功能及在癌症所扮演的角色 15
1-2-4-4 研究動機 16
1-3 實驗材料與方法 25
1-3-1細胞培養 25
1-3-1-1昆蟲細胞的培養 25
1-3-1-2哺乳動物細胞的培養 25
1-3-2重組桿狀病毒的建構與製備 26
1-3-2-1重組桿狀病毒的建構 26
1-3-2-2質體轉染試驗 29
1-3-2-3重組桿狀病毒的放大 29
1-3-2-4超高速離心濃縮桿狀病毒 30
1-3-2-5重組桿狀病毒感染效價的測定 30
1-3-3 桿狀病毒的轉導策略 31
1-3-4 實驗分析方法 31
1-3-4-1即時偵測同步定量聚合酶連鎖反應分析 31
1-3-4-1-1 Total RNA 抽取 32
1-3-4-1-2 cDNA的合成 32
1-3-4-1-3 TaqMan microRNA assy 32
1-3-4-2 細胞微球體形成能力分析 33
1-3-4-3 血管新生試驗分析 33
1-3-4-4 微小RNA微陣列分析 34
1-3-4-6 細胞凋亡分析 34
1-3-4-5 腫瘤細胞及正常細胞共同培養分析桿狀病毒的選擇性 35
1-4 結果 42
1-4-1 分析miR196a於肝癌細胞內源性表現量及病毒功能性分析 42
1-4-2 調控miR196a對於肝癌細胞的癌化能力的影響 43
1-4-2-1細胞形成懸浮球體能力的影響 43
1-4-2-2 調控miR196a對於肝癌細胞促血管新生能力的影響 43
1-4-3 分析癌細胞與正常細胞MicroRNA表現圖譜,用於桿狀病毒合成開關設計 44
1-4-3-1以微小RNA微陣列分析microRNA表達圖譜 44
1-4-3-2 以TaqMan microRNA assay確認MicroRNA的表現趨勢 46
1-4-3-3 分析小鼠肝組織miR126, miR218, miR196a的表現量 46
1-4-3-4 分析調控miR196a表現量對於內生性miR126的影響 47
1-4-4帶有合成開關之桿狀病毒調控外來基因在癌細胞的表現 47
1-4-5 帶有合成開關之桿狀病毒對於肝癌細胞及正常細胞毒殺能力 49
1-4-6 以腫瘤細胞及正常細胞共同培養系統分析桿狀病毒的選擇性 50
1-5 討論 52
Part 2. 應用Cas13調控CHO-K1細胞多重基因表現以增加去岩藻糖醣基化抗體的產量與活性 64
2-1 序論 65
2-2 文獻回顧 68
2-2-1 重組抗體/蛋白表現系統 68
2-2-1-1 哺乳類細胞蛋白表現系統 68
2-2-1-2 CHO細胞蛋白表現系統產量提升的方法 68
2-2-1-2-1 細胞基因工程 68
2-2-1-2-2 蛋白表達質體建構 69
2-2-1-2-3 蛋白生產培養基的開發及製程最佳化 69
2-2-2 CRISPR 編輯系統 70
2-2-2-1 Cas13的介紹及其應用 71
2-2-2-2 應用Cas13d系統進行CHO細胞株改良 72
2-2-2-2-1 LDHA 72
2-2-2-2-2 GFT 73
2-2-2-2-3 DDIT3 74
2-3 實驗方法 81
2-3-1 gRNA spacer的設計 81
2-3-2 質體建構 81
2-3-3 細胞培養、轉染及篩選 82
2-3-4 實驗分析方法 83
2-3-4-1 即時定量聚合酶連鎖反應分析 83
2-3-4-2 植物凝集素凝集敏感性測試分析 84
2-3-4-3 DDIT3細胞內染色分析 84
2-3-4-4 以Anti-Her2 IgG based ELISA分析抗體表現量 84
2-3-4-5 專一蛋白產率分析 (specific production rate, SPR) 85
2-3-4-6 以生化分析儀進行葡萄糖及lactate的測定 85
2-3-4-7 細胞氧化壓力測定 (Reactive Oxygen Specie assay) 85
2-3-4-8 馴化CHO細胞至無血清培養基方式 86
2-3-4-9 批次生產 86
2-3-4-10 抗體純化 87
2-3-4-10 抗體依賴腫瘤細胞毒殺性分析 87
2-3-4-11 細胞團塊形成分析 88
2-4 結果 88
2-4-1 設計Cas13-gRNA模組進行標的基因的調控 88
2-4-2 以Sleeping Beauty系統增強Cas13d-gRNA在CHO細胞的穩定性 89
2-4-3 建立Cas13多標的RNA靶向系統 90
2-4-4以CRISPR/Cas13d進行多標的RNA的編輯對於CHO細胞株的存活率及蛋白產量的影響 91
2-4-5 CRISPR/Cas13d多基因調控對於CHO細胞株生產抗體活性及ROS的影響 92
2-5 討論 93
文獻 108
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