比較SV40驅動和IRES驅動二氫葉酸還原酶的載體在CHO細胞工程中基因放大的差異性

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研究生：	林世昌 Shih-Chang Lin
論文名稱：	比較SV40驅動和IRES驅動二氫葉酸還原酶的載體在CHO細胞工程中基因放大的差異性 Comparing the Difference of Gene Amplification between SV40-driven dhfr and IRES-driven dhfr Vectors in CHO Cell chromosome Engineering
指導教授：	吳夙欽 Suh-Chin Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	58
中文關鍵詞：	基因放大、中國倉鼠卵巢細胞、急性嚴重呼吸道症候群、棘蛋白質、次單位疫苗、二氫葉酸還原脢
外文關鍵詞：	gene amplification, CHO cell, SARS, spike protein, subunit vaccine, dihydrofolate reductase
相關次數：	點閱：1 下載：0
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S蛋白在疫苗的研發上扮演了很重要的角色。已知截短的SARS-CoV TW1 病毒株之棘蛋白─ 「STR2」(包含有三個S蛋白片段：S74-253、S294-739和S1129-1255，分子量為88 kDa)可在哺乳動物細胞中被表現出來，其表現主要為以對Endo H酵素敏感的醣蛋白形式(~115 kDa)。為了建立可穩定大量表現STR2蛋白之CHO細胞，這份研究使用了由兩種不同方式驅動dhfr基因表現的載體：ISID由internal ribosome entry site (IRES)驅動dhfr基因，ISIZ由SV40啟動子驅動dhfr基因。逐步提高Methotrexate (MTX)濃度讓STR2基因數伴隨dhfr基因在染色體中放大以期大量表現STR2蛋白，並比較利用兩種載體產生的基因數放大的差異。這份研究除了篩選出STR2表現量高的細胞株作為生產次單元疫苗的利器，並可作為未來設計可放大載體的參考。
哺乳動物細胞因其有轉譯後修飾作用，可以增加重組蛋白的療效、穩定度等，在生技製藥上常被用來生產重組蛋白，又因CHO細胞表現系統可以利用dihydrofolate reductase (dhfr)基因放大來增加產量，因此最被廣泛使用，這份研究即是利用CHO細胞表現嚴重急性呼吸道症候群冠狀病毒(SARS-CoV)的套膜Spike重組蛋白(S蛋白)。

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated SARS-CoV TW1 S protein, STR2 (88 kDa), carrying three S fragments (S-74-253, S-294-739, and S-1129-1255) could express and show the major form as the Endo H-sensitive (~115 kDa) in CHO cells. To establish stably expressing cell clones, the CHO/dhFr- cells were transfected with two amplifiable vectors, ISID (IRES-driven dhfr) and ISIZ (SV40-driven dhfr), for stepwise MTX selection. The enhancement of the ~115 kDa glycoform generation was observed through gene amplification. After stepwise MTX selection, we can compare the difference of gene amplification between the two vectors in CHO cell chromosome engineering. This study can provide information for development of mammalian cell-based SARS-CoV subunit vaccines, and for amplifiable vectors design to produce recombinant proteins.
Because post-translational modification could improve the efficiency and stability of the recombinant proteins in mammalian cells, mammalian cells are the most common used as host cells to produce biopharmaceuticals. CHO cells, which can amplify target gene following dhfr gene amplification to increase productivity, are used as host cells in this study.

中文摘要……………………………………………………………………………….I
English Abstract……………………………………………………………………...II
致謝..............................................................................................................................III
Content………………………………………………………………………………IV
Figures List………………………………………………………………………….VI

1. Introduction…………………………………………………………………..........1
1.1    Mammalian Cell Expression System    1
1.1.1    Overview of Mammalian Cell Expression System    1
1.1.2    Current Progress in CHO Cell Engineering    4
1.2    CHO/dhFr- Cell Expression System    6
1.2.1    History of dhfr Gene Amplifiction    6
1.2.2    dhfr Gene Amplification in CHO cells    7
1.2.3    Principle of dhfr Gene Amplification    8
1.2.4    Stability of Amplified Genes    10
1.3    Spike Protein of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)    11
1.3.1    History of SARS-CoV    11
1.3.2    SARS-CoV Spike Protein    12
1.3.3    Spike Protein as Target for SARS-CoV Vaccine Development    13
1.4    Goal of the Research    15
2. Materials and Methods…………………………………………………………..17
2.1.    Cell Lines and Mediums    17
2.2.    Expression Vectors Construction    17
2.3.    Polymerase Chain Reaction (PCR)    18
2.4.    Restriction Enzyme Cutting    19
2.4.1    Plasmid Cutting for ligation    19
2.4.2  PCR Product Cutting for Ligation    19
2.4.3  Plasmid Cutting for Selection Correct E. coli Clones    19
2.5.    Ligation    20
2.6.    Transformation    20
2.7.    DNA Concentration Determination    21
2.8.    Transient Transfection by ExGen 500    21
2.9.    SDS-PAGE    22
2.10.    Western Blot    22
2.11.    Lipofectamine 2000 Transfection of CHO/dhFr - cells for Stable Clone Selection    23
2.12.    Selection and Gene Amplification of Clones that Can Stably Express STR2 Protein    24
2.13.    Quantitative Real-Time PCR    25
3. Results…………………………………………………………………………….26
3.1   Transient Expression of STR2 and DHFR    26
3.2    Stable Clone selection and Gene Amplification Through Gradually increasing MTX concentration    26
3.3    Stability of The Amplified Genes    28
3.4    dhfr Gene Copy Number    29
3.5    STR2 expression in the serum-free medium    30
4. Discussion…………………………………………………………………………32
4.1   The Activity of Promoter Driving Zeor was possible to affect selecting stable clones    32
4.2    Glycosylation Patterns of STR2 Produced in CHO/dhFr- Cells    33
4.3    STR2 Expression and Gene Amplification Comparison    35
4.4    Stability of Amplified Genes    36
4.5    STR2 expression in the serum-free medium    36
4.6    Conclusion    37

FIgures……………………………………………………………………………….39
References……………………..…………………………………………………….53

Figures
Figure 1.  Dihydrofolate reductase (DHFR) related metabolism pathway.    39
    Figure 2.  Construction of STR2………………………………………………40
Figure 3.  Construction of amplifiable vectors, pISID and pISIZ.    41
Figure 4.  The flowchart of establishing amplified stable cell clones expressing recombinant proteins in CHO/dhfr- expression system.    42
Figure 5.  STR2 Glycoform    43
Figure 6.  The transient expression of STR2    44
Figure 7.  The transient expression of DHFR    45
Figure 8.  Western blotting of each clones    46
Figure 9.  Results of MTXselection    47
Figure 10.  The STR2 expression of the cells transfected with ISID in the process of MTX selection    48
Figure 11.  The STR2 expression of the cells transfected with ISIZ in the process of MTX selection    49
Figure 12.  STR2 expression of ISID and ISIZ clones comparison    50
Figure 13.  dhfr gene copy number comparison    51
Figure 14.  STR2 expression in the CHO-DHFR (serum-free) medium    52

                                

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