臺灣博碩士論文加值系統

沙利竇邁（Thalidomide）在五零年代末期上市，在當時被視為最安全的鎮定劑。由於作用快、不成癮、無毒性，同時也因對抑制懷孕造成的嘔吐非常有效，使它被廣為使用。但六零年代初期由於會造成畸胎、死胎而遭禁用。進一步瞭解發現，由於其具有光學活性，其中(+)-Thalidomide為鎮定劑的主要成分，而(-)-Thalidomide卻因會抑制新生血管而導致畸胎、死胎。近來隨著癌細胞餓死理論的提出，Thalidomide再度受到重視。由於Thalidomide會抑制腫瘤細胞的血管生成，可使腫瘤細胞自然萎縮消失。
在本實驗中，我們利用接上(±)-Thalidomide的胎牛血清蛋白（Bovine Serum A1bumin, BSA）作為抗原免疫小鼠，以產生對Thalidomide有親和力的抗體，在血清的ELISA （Enzyme-Linked Immunosorbent Assay）效價達到一定值後，藉本實驗室所發展之噬菌體單鏈抗體展示基因庫技術，依下列流程：將免疫後小鼠脾臟細胞的抗體重鍊可變區VH、抗體輕鍊可變區VL之mRNA基因反轉錄後，藉由聚合酵素連鎖反應（PCR）分別將VH、VL之cDNA放大，並藉SOE-PCR（Splicing by overlap extension PCR）將兩者隨機組裝成單鏈抗體以構築一個噬菌體抗體展示基因庫。在得到大量噬菌體抗體後藉由對Thalidomide的親和力反覆挑選出分別能夠辨識(+)-Thalidomide與(-)-Thalidomide的噬菌體抗體，並以競爭型ELISA進行確認。本實驗最後得到分別對(+)-Thalidomide與(-)-Thalidomide有親和力之噬菌體抗體。

Thalidomide was first introduced in the 1950s as a sedative drug. Because it was deemed to be safe, it was prescribed for nausea and insomnia in pregnant women. However, it was found to cause the severe birth defects in children whose mothers had taken the drug in their first trimester of pregnancy. This was a terrible tragedy. Deepgoing research realized that one of thalidomide enantiomer possesses the desired therapeutic effect of quelling nausea, but its mirror compound produces fetal deformities.
In this work, we synthesis thalidomide conjugated with BSA as an immunogen in order to produce related antibodies. After immunization, RT-PCR was performed and a series of primers was used to amplify splenic VH/VL genes. Then, amplified VH/VL were assembled to construct the antibody phage display library against thalidomide. The constructed antibody phage display library was being selected against immunogen after comfirming the diversity by BstNI restriction enzyme fingerprinting analysis. After three rounds of selection, we have isolated two phage clones with weak affinity against (+) or (-) thalidomide individually. Colony PCR and western blotting analysis showed these two clones bear full-length of scFv DNA fragment and are able to express scFv. Competitive ELISA assay also confirmed the specificity of these two scFvs against (+)-thalidomide or (-)-thalidomide respectively.

壹、緒論 1
一、鏡像異構物（enantiomer） 1
二、沙利竇邁（Thalidomide） 3
三、Thalidomide的藥理學 4
四、Thalidomide的新醫學用途 4
五、單鏈單株抗體（Monoclonal scFv） 5
六、噬菌體展示系統（phage display system） 8
七、研究動機 9
貳、實驗流程 12
參、實驗材料與方法 14
一、材料 14
二、方法 16
1.製備Thalidomide抗原 17
2.Thalidomide抗原確認與定量 17
3.免疫 BALB/ c ByJ (female) mice 17
4.間接酵素連結免疫測定法 17
5.Phage antibodies ELISA assay 18
6.Competitive Phage antibodies ELISA assay 19
7.Total RNA萃取 20
8.反轉錄反應（Reverse transcription） 21
9.單鏈抗體基因庫構築 21
10.DNA的洋菜膠體電泳分析、純化與定量 24
11.限制酶切割 26
12.製備質體 26
13.scFv DNA與質體接合反應 27
14.熱休克勝任細胞之製備 27
15.電穿孔勝任細胞之製備 28
16.熱休克轉型作用 28
17.電穿孔轉型作用 27
18.自大腸桿菌中產生噬菌體抗體 28
19.篩選具有親合力之噬菌體抗體 30
20.噬菌體基因庫BstNI限制酶圖譜分析法 30
21.單鏈抗體的表現 31
22.蛋白質SDS-PAGE電泳膠體分析蛋白 32
23.西方轉漬法(Western blotting) 32
肆、結果與討論 34
一、Thaidomide抗原合成與分析 34
二、小鼠血清抗Thalidomide之IgG的效價分析 36
三、製備抗Thalidomide之單鏈抗體基因庫 37
四、篩選具有親合力之噬菌體抗體 40
五、西方點墨法（western blotting） 42
伍、參考文獻 44
陸、附錄 47
附錄一：本實驗使用引子序列 47
附錄二：SDS-Page電泳膠體配置 51
附錄三：常用緩衝溶液與培養基 52
附錄四：縮寫字對照表 53

(1)Crosby J. (1991) Synthesis of optically active compounds: A large scale perspective, Tetrahedron 47:4789-4846.
(2)Mellin G. W, Katzenstein M. (1962) The saga of thalidomide: neuropathy to embryopathy with case reports and congenital abnormalities. N. Engl. J. Med. 23:1184-90.
(3)Peuckmann V, Fish M, Bruera E. (2000) Potential novel uses of thalidomide Drugs.60:273-92.
(4)Szeinberg A, Sheba C. (1968) Pharmacogenetics. Israel. J. Med. Sci. 4:488.
(5)O''Reilly MS,Boehm T, Shing Y, Fukai N, Vasios G., Lane WS, Flynn E, Birkhead JR, Olsen BR, and Folkman J. (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, no. 2:277.
(6)Boehm T, Folkman J, Browder T, and O''Reilly MS. (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, no. 6658:404.
(7)Sands BE, Podolsky DK. (1999) New Life in a sleeper: Thalidomide and Crohn''s disease. Gastroenterology. 117:1485-8.
(8)Radomsky CL, Levine N. (2001) Thalidomide. Dermatol Clin.19:87-103.
(9)Calderon P, Anzilotti M, Phelps R. (1997) Thalidomide in dermatology: New indications for an old drug. Int J Dermatol. 36:881-7.
(10)Verheul HM, Panigrahy D, Yuan J, D''Amato RJ. (1999) Combination therapy with thalidomide and sulindac inhibits tumor growth in rabbits. Br J Cancer. 79:114-8.
(11)Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, et al. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci.. USA. 85:5879-5883.
(12)McCafferty J, Griffiths AD, Winter G, Chiswell DJ. (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 348:552-554.
(13)Foy BD, Killeen GF, Frohn RH, Impoinvil D, Williams A, Beier JC. (2002) Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens. J Immunol Methods 261:73-83.
(14)Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I, Winter G, Karsten U. (2002) Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 315:1087-1097
(15)Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M, Crawford RS, Weiner LM, Marks C, Marks JD. (1996) Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Bio 263:551-567.
(16)Daugherty PS, Chen G, Iverson BL, Georgiou G. (2000) Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proc Natl Acad Sci U S A 97:2029-2034.
(17)Chowdhury PS, Pastan I. (1999) Improving antibody affinity by mimicking somatic hypermutation in vitro. Nat Biotechnol 17:568-572.
(18)Vitaliti A, Wittmer M, Steiner R, Wyder L, Neri D, Klemenz R. (2000) Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor. Cancer Res. 60:4311-4314.
(19)Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD. ( 2000 ) Selection of tumor-specific internalizing human antibodies from phage libraries. J Mol Biol 301:1149-1161.
(20)Bera TK, Kennedy PE, Berger EA, Barbas CF 3rd, Pastan I. (1998) Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein. Mol Med. 4:384-391.
(21)DeNardo SJ, DeNardo GL, DeNardo DG, Xiong CY, Shi XB, Winthrop MD, Kroger LA, Carter P. (1999) Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics. Clin Cancer Res 5: 3213s-3218s.
(22)Kojima K. (2001) High-performance immunoaffinity chromatography, an immunoaffinity membrane for selective removal of plasma components, and safety evaluation of the latter system J. Biochem. Biophys. Method.s 49:241-251.
(23)Sang BL, David TM, Lacramioara T, Tarja K. Nevanen, HS, and Charles RM. (2002) Antibody-Based Bio-Nanotube Membranes for Enantiomeric Drug Separations Science. 296:2198-2200.
(24)Winthrop MD, Denardo GL, Denardo SJ. (2000) Antibody phage display applications for nuclear medicine imaging and therapy. Q J Nucl Med. 44:284-295.