齒舌蘭輪斑病毒(Odontoglossum ringsport virus，ORSV )是感染蘭科植物重要的病毒，除了影響植株生長外，亦會造成蘭花品質下降，因此，一般用ELISA檢測蘭花ORSV病毒。本研究為構築ORSV phage display library 從中篩選產生scFv 抗體。
本試驗依據ORSV的鞘蛋白(coat protein )基因序列設計引子(primers)，將鞘蛋白基因選殖至質體表現系統，由GST(gutathione S-transferase)融合蛋白中純化得到ORSV鞘蛋白，再將鞘蛋白注射至老鼠體內產生免疫反應後，抽取老鼠脾臟細胞的total RNA，進行反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction, RT-PCR)，將ORSV的抗體重鏈及輕鏈反轉錄成cDNA，利用聚合酵素反應法(polymerase chain reaction，PCR)分別選殖出重鏈及輕鏈的變異區片段，並藉由重疊延伸的基因裁切法(gene splicing by overlap extension)構築出ORSV- scFv DNA，利用phage display system構築ORSV phage display library，再用ORSV鞘蛋白進行親合性的篩選(bio-panning) 三次，篩選到C10、C11、D5、F10等4個重組噬菌體株，這些菌株產生的scFv antibody 對ORSV有專一性的親合反應。以scFv antibody檢查感染ORSV 的蝴蝶蘭葉片萃取液，結果顯示用scFv antibody ELISA 的檢測的OD 405比ORSV多株抗體弱，但最低病毒檢測濃度為10 ng/μl與ORSV 多株抗體的檢測極限相似。本試驗得到的scFv antibody，其檢測靈敏性相當於動物免疫多株抗體，未來希望能利用phage display 技術量產具專一性和靈敏度高的scFv 抗體，應用於病毒的檢測。

Odontoglossum ringspot virus (ORSV) is one of the commonest viral pathogens of the cultivated orchids. Infected plants appear unhealthy and may produce low quality flowers. In this experiment a set of ORSV-specific oligonucleotide primers were designed from the region of the coat protein (CP) gene of ORSV. The ORSV CP gene was cloned into the protein expression bacterial plasmid vector of the gutathione S-transferase（GST）fusion protein expression system. The recombinant ORSV CP was injected into mouse to induce immune response of the animal. The cDNAs of VH and VL of ORSV antibody genes were obtained by using reverse transcription polymerase chain reaction from the total the RNAs that were extracted from the spleen cells of immunized mouse. ScFv（single-chain variable fragment）library of ORSV were constructed with gene splicing by overlap extension. Thirty seven scFvs were selected from ORSV-scFv library following three rounds of affinity selection with ORSV CP as an antigen that was expressed in bacteria. Four scFv antibody have specific binding reaction against ORSV CP was selected. Comparing the sensitivity between scFv antibodies and ORSV polyclonal antibody tisted in enzyme linked immunosorbent assay (ELISA) to detect ORSV in leaf extracts of diseased Phalaenopsis plants. Unfortunately, the affinity between scFv antibody and ORSV was weaker than that between polyclonal antibody to the same antigen. The scFv antibodies and polyclonal antibody appeared to have similar sensitivity limit as low as 10 ng/μl. The results highlight the potential of applying the scFv antibodies in the diagnosis of virus diseases.

目 錄
中文摘要………………………………………………………………………..i
英文摘要……………………………………………………………………….ii
目錄…………………………………………………………………………....iv
圖目次………………………………………………………………………...vii
附錄目次…………………………………………………………………..…...x
壹、前言……………………………………………………………………….……..1
貳、前人研究…………………………………………………….…………….4
參、材料與方法………………………………………………………………15
一、 齒舌蘭輪點病毒鞘蛋白基因片段的選殖
(一) ORSV病毒RNA的純化……………………………………………15
(二)反轉錄聚合酶連鎖反應(Reverse Transcription Polymerase Chain
Reaction, RT-PCR)……………………………………………….…..16
(三) DNA電泳膠體分析……………………………………………….…17
(四) ORSV鞘蛋白基因片段的純化……………………………………...18
(五) ORSV鞘蛋白基因片段之選殖（cloning）…………………………19
(六)定序………………………………………………………………..….22
二、 齒舌蘭輪點病毒鞘蛋白(coat protein)的表現與純化
(一)GST-ORSV 鞘蛋白融合蛋白質的表現……………………………..23
(二)ORSV 鞘蛋白的純化………………………………………….…….23
(三)聚丙醯氨凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel
electrophoresis，SDS- PAGE) …………………………….………..24
(四)西方墨點分析法 (Western blotting)…………………………………25
三、 齒舌蘭輪點病毒(Odontoglossum ringspot tobamovirus, ORSV) scFv
antibody library的構築與表現
(一) ORSV鞘蛋白免疫BALB/cByJ (female)老鼠………………………27
(二)間接酵素連結免疫測定法(indirect enzyme-linked immunosorbent
assay, ELISA )……………………………………………………...27
(三)西方墨點法(western blotting)檢測老鼠產生免疫反應……….….…28
(四)抽取免疫BALB/cByJ(female)老鼠脾臟細胞(spleen cell)的total
RNA………………………………………………………………...28
(五)抗體重鏈/輕鏈PCR放大反應與scFv antibody組裝與表現……….29
四、篩選(biopanning)和soluble scFv 抗體的製備
(一)誘導TG1釋出重組噬菌體…………………………………………...35
(二)scFv 重組噬菌體的篩選(biopanning)…………………………….....35
(三)用ELISA篩選 phage-displayed ORSV scFv抗體………………….38
(四)可溶性抗體片段(soluble scFv antibody)的製備…………………….38
(五)scFv antibody 的純化………………………………………………...40
(六)西方墨點法(Western blot) 分析scFv抗體的表現………………….41
(七)定序分析……………………………………………………………...42
(八)soluble scFv 抗體與蘭花病毒萃取液、ORSV病毒顆粒之間的親合
力檢測……………………………………………………………........42
(九)scFv 抗體濃度定量分析……………………………………………..43
肆、結果………………………………………………………………………44
一、齒舌蘭輪點病毒的選殖…………………………………………………44
二、齒舌蘭輪點病毒鞘蛋白的表現與純化………………………………….49
三、齒舌蘭輪點病毒(Odontoglossum ringspot tobamovirus, ORSV) scFv
DNA library的構築與表現……………………………………………...52
四、scFv 重組噬菌體的篩選(biopanning)與可溶性scFv antibodies的
製備……………………………………………………………………...60
伍、討論……………………………………………………………………….74
陸、參考文獻…………………………………………………………………85

洪健恆。2000。抗warfarin之抗體及單鏈抗體之製備、分析與應用。國立中興大學/生物化學研究所106頁。
劉賢玲。2000。臺灣蝴蝶蘭之病毒病因研究。國立台灣大學植物病理研究所碩士論文61頁。
張清安、曾雅詩、陳金枝、鄧汀欽。1999。應用聚合酶連鎖反應及核酸探針雜配法偵測齒舌蘭輪斑病毒。植物病理學會刊。8：29-36。
張清安。1996。蘭花病毒病之特性與防治。農業世界。151：14-20。
Arunasalam, G., and M. N. Pearson. 1989. ELISA detection of
odontoglossum ringspot virus in mature plants and protocorms of Cymbidium orchids :Potential solutions to problems of sample preparation time and low virus concentration. J. Phytopathol. 126:160-166.
Barry, K. J. S. Hu, A. R. Kuehnle, and N. Sughii. 1996. Sequence virus and odontoglossum ringspot virus in Hawaiian orchids. J. Phytopathol. 144:179-186.
Benhar, I. 2001. Biotechnological applications of phage and cell display. Biotechnology Advances 19:1-33.
Boonham, N., I. Barker. 1998. Strain specific recombinant antibodies to Potato virus Y potyvirus. J. Virol. Methods 74:193-199.
Chae, J. S., J. K. Choi, H. T. Lim, S. H. Cha. 2000. Generation of a murine single chain Fv (scFv) antibody specific for Cucumber Mosaic Virus (CMV) using a phage display library. Mol. Cell 11:7-12.
Chng, C. G., S. M. Wong, P. H. Mahtani, C. S. Loh, C. J. Goh, M. C. Kao, M. C. Chung. 1996. The complete sequence of a Singapore isolate of odontoglossum virus and comparison with other tobamoviruses. Gene 171:155-161.
Deng, X. K., L. A. Nesbit, and K. J. Morrow. 2003. Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system. Clin. Diagn. Lab. Immunol 10:587-595.
Eun, A. J., and S. M. Wong. 1999. Detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus using immuno-capillary zone electrophoresis. Phytopathology 89:522-528.
Eun, A. J., and S. M. Wong. 2000. Simultaneous quantitation of two orchid viruses via the TaqMan real time RT-PCR. J. Virol. Methods 87:151-160.
Eun, A. J., and S. M. Wong. 2000 b. Molecular beacons: a new approach to plant virus detection. Phytopathology 90:269-275.
Gough, K. C., W. Cockburn, G. C. Whitelam. 1999. Selection of phage-display peptides that bind to cucumber mosaic virus coat protein. Journal of virological methods 79:169-180.
Griep, R. A., C. V. Twisk, and A. Schots. 1999. Selection of beet necrotic
yellow vein virus specifis single-chain Fv antibodies from a semi-synthetic combinatorial antibody library. European journal of plant pathology 105:147-156.
Griep, R. A., M. Prins, C. V. Twisk, H. J. Keller, F. J. Kerschbaumer, R. Kormelink, R. W. Goldbach, and A. Schots. 2002. Application of phage display in selection Tomato spotted wilt virus-specific single chain Fvs for sensitive diagnosis in ELISA. Phytopathology 90:183-190.

Griffiths, A. D., and A. R. Duncan. 1998. Strategies for selection of
antibodies by phage display. Current opinion in Biotechnology
9:102-108.
Harper, K., R. J. Kerschbaumer, A. Ziegler, S. M. Macintosh., G. H. Cowan, G. Himmler, M. A. Mayo, L. Torrance. 1997. A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA. J. Virol. Methods 63:237-242.
Harper, K., R. L. Toth, M. A. Mayo, L. Torrance. 1999. Properties of a panel of single chain variable fragments against Potato leafroll 6irus obtained from two phage display libraries. Journal of Virological Methods 81:159-168.
Hassan, M. E, W. E. Highsmith. 2002. Phage display technology: clinical applications and recent innovations. Clinical Biochemistry 35:425-445.
Hoogenboom, H. R., A. P. Bruine, S. E. Hufton, R. M. Hoet, J. W. Arends, R. C. Roovers, 1998. Antibody phage display technology and its applications. Immunotechnology 4:1-20.
Hull, R. 1984. Caulimovirus group. CAB/AAB Descriptions of Plant Viruses, no. 295.
Hu, J. S., S. Ferreira, and M. Wang. 1993 Detection of cymbidium mosaic virus, odontoglossum ringspot virus, tomoto spotted virus, and potyviruses infection orchids in Hawaii. Plant Dis. 77:464-468.
Hu, W. W., and S. M. Wong. 1998. The use of DIG-labeled cRNA probes for the detection of cymbidium mosaic potexvirus (CyMV) and odontoglossum ringspot tobamovirus (ORSV) in orchids. J. Virol. Methods 70:193-199.

Hu, W. W., S. M. Wong, C. S. Loh, and C. J. Goh. 1998. Synergism in replocition of cymbidium mosaic potexvirus (CyMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts. Arch Virol. 143:1265-1275.
Inouye, N. 1977. Serological diagnosis methods for Cymbidium mosaic mosaic virus and odontoglossum ringspot viruse in orchids. Nogaku Kenkyu 56:1-13.
Inouye, N. 1996. A virus disease of Cymbidium caused by odontoglossum ringspot virus. Ber Ohara Institutes 149-159
Jensen, D. D., A. H. Gold. 1951. A virus ringspot of odontoglossum orchid: symptoms,transmission, and electron microscopy. Phytopathology 41: 648-653.
Jes´us, R. D., M. Vicente, P. M. Gaspar, B. Javier. 2004. Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display. Journal of Virological Methods 121:231-238.
Johnson, S., and R. E. Bird. 1991. Construction of single-chain Fv derivatives monoclonal antibodies and their production in Escherichia coli. Methods in Enzymology 203: 88-98.
Jun, P. Y., J. H. Ko, Y. P. Qi. 2004. Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library. Thrombosis research 114:205-211.
Kang, A. S., C. F. Barbas, K. D. Janda, S. J. Benkovic, R. A. Lemer. 1991. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci USA 88:4363-4366.
Kevin, C. G., C. William, G. C. Whitelam. 1999. Selection of phage-display peptides that bind to cucumber mosaic virus coat protein. Journal of Virological Methods 79:169-180.
Lim, S. T., S. M. Wong, C. Y. Yeong, S. C. Lee, and C. J. Goh. 1993. Rapid detection of cymbidium mosaic virus by the polymerase chain reaction. J. Virol. Methods 41:37-46.
Mead, D. A., B. Kemper. 1988. Chimeric single-stranded DNA phage-plasmid cloning vectors. Biotechnology 10:85-102.
Michael, H., E. Maiss., H. J. Jacobsen., T. Reinard. 2002. The production of a genus-specific recombinant antibody(scFv) using a recombinant potyvirus protease. Journal of Virological Methods 106:225-233.
Murakishi, H. H. 1958. Serological and morphological relationship among orchid viruses. Phytopathology 48: 137-140.
Nissim, A., H. R. Hoogenboom, I. M. Tomlinson, G. Flynn, C. Midgley, D. Lane, G. Winter. 1994. Antibody fragments from a single pot phage display library as immunological reagents. EMBO J. 13:692-698.
Okamoto, T., Y. Mukai, Y. Yoshioka, H. Shibata, M. Kawamura, Y. Yamamoto, S. Nakagawa, H. Kamada, T. Hayakawa, T. Mayumi, Y. Tsutsumi. 2004. Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen. Biochemical and Biophysical Research Communications 323:583-591.
Pearson, M. N, J. S. Cole. 1986. The effects of cymbidium mosaic virus and odontoglossum ringspot virus on the growth of Cymbidium orchids. J. Phytopathol 117: 193-197.
Pearson, M. N., J. S. Cole. 1991. Further observations on the effects of cymbidiummo saic virus and odontoglossum ringspot virus on the growth of Cymbidium orchids. J. Phytopathol 131:193-198.
Ryu, K. H. and W. M. Park. 1995. The complete nucleotide sequence and genome organization of odontoglossum ringspot tobamovirus RNA. Arch. Virol. 140:1577-1587.
Ryu, K. H., and W. M. Park. 1995. Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification. FEMS Microbiol. Lett. 133:265-269.
Saldarelli, P., H. Keller, M. Dell’Orco., A. Schots, V. Elicio, A. Minafra. 2005. Isolation of recombinant antibodies (scFv) to grapevine virus B. Journal of Virological Methods 124:191-195.
Sachdev, S. S. 2000. Phage display in pharmaceutical biotechnology. Current Opinion in Biotechnology 11:610-616.
Sachdev, S. S., 2001. Engineering M13 for phage display Biomolecular Engineering 18:57-63.
Scott, J. K., G. P. Smith. 1990. Searching for peptide ligands with an epitope library. Science 249:386-390.
Seoh, M. L., S. M. Wong, and L. Zhang. 1998. Simultaneous TD/RT PCR detection of cymbidium mosaic potexvirus and odontoglossum ringspot tobamovirus with a single pair of primers. J. Virol. Methods 72:197-204.
Sheets, M. D., P. Amersdorfer, R. Finnern, P. Sargent, E. Lindqvist, R. Schier, G. Hemingsen, C. Wong, J. C. Gerhart, and J. D. Marks. 1998. Efficient construction of a large non-immune phage antibody library: The production of high-affinity single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. U.S.A. 95:6157-6162.
Susi, P., A. Ziegler, L. Torrance. 1998. Selection of single chain variable fragment antibodies to blackcurrant reversion associated virus from a synthetic phage display library. Phytopathology 88:230-233.
Tan, S. W. L., S. M. Wong, R. M. Kini. 2000. Rapid simultaneous
detection of two orchid viruses using LC- and:or MALDI-mass
spectrometry. J. Virol. Methods 85:93-99.
Tessmann, K., A. Erhardt, D. Haussinger and T. Heinges. 2002. Cloning and molecular characterization of human high affinity antibody fragments against hepatitis Cvirus NS3 helicase. Journal of Virological Methods 103:75-88.
Torrance, L. 1992. Serological methods to detect plant viruses: production and use of monoclonal antibodies. In: Duncan, J.M., Torrance, L. (Eds.), Techniques for the Rapid Detection of Plant Pathogens. Blackwell, Oxford, pp. 7–33.
Uhde, K., R. J. Kerschbaumer, R. Koenig1, S. Hirschl, O. Lemaire, N. Boonham, W. Roake, and G. Himmler. 2000. Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries. Arch Virol 145:179-185.
Vaughan, T. J., A. J. Williams, K. Pritchard, J. K. Osbourn, A. R. Pope, J.C. Earnshaw, J. McCafferty, R. A. Hodits, J. Wilton, and K. S. Johnson. 1996. Human antibodies with sub-nanomolar affinities isolated from a large nonimmunized phage display library. Nature Biotechnol 14:309-314.
Wittrup, K. D. 1999. Phage on display. Trends Biotechnol 17:423-424.
Wisler, G. C., F. W. Zettler, L. Mu, 1987. Virus infection of Vanilla and other orchids in French Polynesia. Plant Dis. 71:1125-1129.
Wisler, G. C., F. W. Zettler, and T. J. Sheehan, 1983. A diagnostic technique developed for orchid growers to detect cymbidium mosaic and odontoglossum ringspot viruses. Am. Orchid Soc. Bull 52:255-261.
Wong, S. M., C. G. Chng, Y. H. Lee, K. Tan, F. W. Zettler. 1994. Incidence of cymbidium mosaic and odontoglossum ringspot viruses and their significance in orchid cultivation in Singapore. Crop Protect 13:235-239..
Wong, S M., S. H. Lim, and C. G. Chang. 1994. Detection of odotoglossum ringspot virus and tobacco mosaic virus with homologoes and geterologous antigen antibody combinations. Plant Dis 12:107-116.
Yu, H. H. and S. M. Wong. 1998. A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene. Arch Virol 143:163-171.
Ziegler, A., L. Torrance., S. M. Macintosh., G. H. Cowan, M. A. Mayo. 1995. Cucumber mosaic cucumovirus antibodies from a synthetic phage display library. Virology 214:235-238.