臺灣博碩士論文加值系統

抗體可以廣泛地應用在診斷、治療、實驗研究。目前有兩種產生高親和
力單株抗體的技術，一種是用細胞融合技術將B 細胞與骨髓瘤細胞融合成融
合瘤細胞 (hybridoma) 產生出單株抗體，另一種是利用轉殖鼠產生人類抗體，
第三種是將抗體基因表現在嗜菌體 (phage)的外殼蛋白 (capsid)。儘管融合瘤
細胞是主要產生單株抗體的技術，但這項技術還是存在許多問題。例如，融
合瘤細胞來自於老鼠，由於抗原性而無法直接應用在人體。因此對於科學家
來說嗜菌體抗體呈現系統 (phage antibody display system) 更具意義。我們試
著利用嗜菌體呈現系統建構出人類單鏈抗體基因庫。首先我們先從290 位捐
贈者血液中分離出淋巴球的mRNA 進行反轉錄聚合��連鎖反應產生重鏈和輕
鏈的cDNA。重鏈和輕鏈cDNA 片段利用31 種不同的引子 (primer)進行聚合
��連鎖反應產生重鏈和輕鏈變異區。將這些重鏈和輕鏈變異區利用重疊聚合
�◆簿�連鎖反應和52 種不同的連接子 (linker) 產生單鏈抗體 (scFv) 基因庫。
這52 種不同的連接子皆含有15 個胺基酸 (Gly4Ser)3，在末端帶有不同序列可
以和用其他不同的引子合成的 VH 基因和VL 基因重疊。純化過的 scFv 片段和
質體pIGT5 接合後，利用電穿孔法將已接合scFv 片段送入大腸桿菌 HB2151
中。為了增加那些表現單鏈抗體的嗜菌體的親和力，因此我們建立了製備了
質體pIGT5 和建立了Ex-phage 系統。質體pIGT5 在scFv-III 融合蛋白之間擁
有胰蛋白�� (Trypsin) 和腸激�� (Enterokinase) 兩個切位。Ex-phage 的pIII 基
因前有一個amber 終止密碼，在無法抑制amber 終止密碼的大腸桿菌株
HB2151 中不會表現pIII，使表現在嗜菌體外的pIII 全部被scFv-pIII 融合蛋白
替代。因此接上抗原的嗜菌體可以加入胰蛋白�〝M腸激��切除單鏈抗體，再
去感染大腸桿菌HB2151 做下一次的篩選。在完成單鏈抗體基因庫後，我們
希望可以應用這單鏈抗體基因庫搜尋到特定的醣抗體。

Antibodies are applied not only for diagnosis, but also in research and
clinical treatments. In order to generate high-affinity monoclonal antibodies, three
techniques have been developed including hybridoma engineering, transgenic mice
and phage-displayed antibody libraries. Although the hybridoma technology is the
major tool for monoclonal antibody production, it still has some problems. For
example, the methodology cannot be curable directly in human disease. Hence, the
phage antibody display system has become more attractive to scientists. We
attempted to develop a na��ve human single chain antibody (scFv) library by phage
display system. We first extracted total RNA from the leukocytes of peripheral
blood from 290 volunteers. The heavy chain and light chain cDNA fragments were
synthesized by reverse transcription reaction. Thirty-one forward and reverse
primers were used for the cloning of heavy chain variable regions (VH) and light
chain variable regions (VL) fragments from the cDNA fragments. The cloned heavy
and light chain variable regions were amplified and linked with linkers by
overlapping PCR. The linkers encoded the 15 amino acids (Gly4Ser)3 that have
overhangs of perfect complementarity with VH and VL genes. The purified scFv
fragments were ligated to the phagemid pIGT5 for electroporation into Escherichia
coli HB2151 competent cells. In order to increase the binding avidity of scFv
during bio-paining, we had constructed pIGT5 phagemid which expressed trypsin
and enterokinase cutting site between pIII and scFv. We also established Ex-phage
in which the pIII protein could not be synthesized in non-amber suppressed strain
owing to the amber codon before pIII. Hence, the binding phage can be released
after enzymatic digestion and amplified for next paining. After the library
constructed, we will apply this antibody library to discover new carbohydrate
antibodies.

Contents
Contents ........................................................................................................ I
Figure contents .......................................................................................... III
摘要 ............................................................................................................... 1
Abstract ........................................................................................................ 2
致謝 ............................................................................................................... 3
Introduction .................................................................................................. 4
Phage Display ............................................................................................. 4
M13 phage .................................................................................................. 4
Application of phage display ...................................................................... 5
Amber codon .............................................................................................. 6
Biopanning ................................................................................................. 7
Phage titer analysis ..................................................................................... 8
Antibody ..................................................................................................... 8
Carbohydrate antibodies ........................................................................... 10
Study Design ............................................................................................... 11
Materials and Methods .............................................................................. 12
Bacteria Strain .......................................................................................... 12
Peripheral Blood Samples ........................................................................ 12
RNA Extraction ........................................................................................ 13
Reverse Transcription Reaction (RT) ....................................................... 13
Polymerase Chain Reaction (PCR) of variable region .............................. 13
Polymerase Chain Reaction (PCR) of scFv linker DNA .......................... 14
Overlap PCR ............................................................................................ 14
Construction of Plasmid pIGT5 ................................................................ 15
Mutagenesis of helper phage .................................................................... 16
Electroporation and Rescue of phage display library ............................... 16
Phage titration ........................................................................................... 17
Western Blot ............................................................................................. 17
Biopanning ............................................................................................... 17
Table.1 ...................................................................................................... 19
Results ......................................................................................................... 23
Generation of pIGT5 phagemid for display of scFv fragment .................. 23
Construction of scFv gene repertoires ...................................................... 23
Ex-phage production ................................................................................ 24
Phage rescue and analysis......................................................................... 24
Phage peptide selection for glycan ........................................................... 25
Discussion ................................................................................................... 26
Conclusion .................................................................................................. 29
Reference .................................................................................................... 30
Figures ........................................................................................................ 39
自述 ............................................................................................................. 47

Figure contents
Figure 1. Construction of pIGT5 phagemid ............................................ 39
Figure 2. PCR amplification of human variable heavy (VH) and variable
light (VL). .................................................................................................... 40
Figure 3. PCR amplification of scFv linker DNA. ................................... 42
Figure 4. Assembly of scFv repertoires by overlapping PCR reaction. . 43
Figure 5. Mutagenesis of Ex-phage genome. ........................................... 44
Figure 6. The effect of Ex-phage packing on scFv displayed on
recombinant phage. ................................................................................... 45
Figure 7. Alignment of phage-displayed peptide sequences selected for
Lex and GlcNAc. ........................................................................................ 46

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