臺灣博碩士論文加值系統

喜姆比蘭嵌紋病毒（Cymbidium mosaic virus，CyMV）一直是蘭花栽培者最主要的障礙之一，除了會影響蘭花的生長外，還會造成感染的蘭花品質下降。木瓜輪點病毒（Papaya ringspot virus，PRSV）為木瓜在生長期間最嚴重的病害之一。由喜姆比蘭嵌紋病毒的抗體基因cDNA（CyMV-H 10-1、CyMV-L 23）及木瓜輪點病毒的抗體基因cDNA（PRSV-H 10-9、PRSV-L 3-8）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scFv-CyMV及scFv-PRSV單鏈抗體基因。經核苷酸序列定序分析，得知scFv-CyMV有723個核苷酸，可轉譯成241個胺基酸，而scFv-PRSV有726個核苷酸，可轉譯成242個胺基酸。為了評估scFv-CyMV及scFv-PRSV單鏈抗體蛋白質的表現情形，構築CyMV及PRSV單鏈抗體基因蛋白質表現質體pCANTAB-CyMV及pCANTAB-PRSV，並利用噬菌體表現重組抗體系統分別轉殖至E. coli TG1，並培養出570個及約1242個菌落。由非直接酵素結合抗體檢定法（indirect ELISA）篩選出12個scFv-CyMV（編號C6、C7、C10、C11、C12、C13、C22、C28、C37、C38、C50及C56）及5個scFv-PRSV（P4、P22、P23、P42及P46）呈正反應菌株來進行聚合酶連鎖反應分析及酵素切割。scFv-CyMV及scFv-PRSV聚合酶連鎖反應結果分別為744及747個核苷酸片段，而酵素切割結果兩者皆約1500個核苷酸片段。選取編號為C6、C10、C11的scFv-CyMV轉殖入E. coli HB2151並利用indirect ELISA分析可溶性抗體在細菌的supernatant、periplasmic extract及whole cell extract的表現濃度，結果以位於supernatant最多。為了進行抗體基因轉殖植物和抗體基因蛋白質大量表現，利用聚合酶連鎖反應技術分別完成CyMV和PRSV單鏈抗體基因用來轉殖植物重組質體pBin-19之基因片段，及CyMV和PRSV單鏈抗體基因蛋白質大量表現重組質體pIVEX之基因片段。

Cymbidium mosaic virus（CyMV） is a major limiting factor for orchid plants. Infected plants are unhealthy and produce lower quality flowers. Papaya ringspot virus（PRSV）is a significant pathogen in papaya. The cDNAs of VH and VL of CyMV and PRSV antibody genes were obtained by using polymerase chain reaction. ScFv（single-chain variable fragment）of CyMV and PRSV were constructed by gene splicing by overlap extension (gene SOEing). Results of nucleotide sequences analysis revealed 723 and 726 nucleotides encoding for 241 and 242 amino acids, respectively, for scFv-CyMV and scFv-PRSV. In order to express scFv-CyMV and scFv-PRSV antibody, pCANTAB-CyMV and pCANTAB-PRSV were constructed for transformation into E. cloi TG1 and 570 and 1242 of colonies, respectively, from CyMV and PRSV phage display system were obtained. Twelve scFv-CyMV and five scFv-PRSV colonies were selected with positive reactions in indirect ELISA. 744 and 747 nucleotides fragments of scFv-CyMV and scFv-PRSV, respectively, were cloned from those positively selected colonies by PCR with specific primers. However, two of 1500 nucleotide fragments were obtained for scFv-CyMV and scFv-PRSV, respectively, by using SfiΙand NotΙ enzyme digestion. C6, C10 and C11 of scFv-CyMV were selected for transformation into E. coli HB2151 for monitoring the locations of scFv-CyMV antibody in E. coli cell including supernatant, periplasm and cytosol. The result showed that scFv-CyMV antibody was most aboundant in supernatant. ScFv-CyMV and scFv-PRSV nucleotide fragments were also cloned by PCR with specific primers for inserting into pBin-19 use for transformation of plants and pIVEX-2.4a use for large scale protein expression.

中文摘要……………………………………………………….……..… I
英文摘要………………………………………….………………… III
壹、前言與前人之研究…………………………………………..…….1
貳、材料與方法…………………………………………..………19
一、抗體基因來源…………………………………………19
二、質體DNA的小量抽取……………………………….19
三、單鏈抗體基因之製備………………………….……..20
(一)確定重鏈及輕鏈的變異區位置………………….20
(二)核酸引子之設計………..………………………….21
(三)聚合酶連鎖反應………………………………..….22
(四)聚合酶連鎖反應產物之電泳膠體分析..………………27
(五)DNA片段的回收與純化….…………………………...27
(六)重疊延伸的基因裁切法……………………………….28
四、單鏈抗體核酸序列之譯讀………………………….…….29
五、單鏈抗體核酸序列之分析………………………….……….32
六、表現單鏈抗體基因蛋白質質體之構築………….……….32
(一)酵素切割………….………………………………..32
(二)黏接反應…………………………………………...33
七、轉殖反應…………………………….………….……….33
(一)勝任細胞………….………………………………..33
(二)轉殖反應…………………………………………...36
八、重組嗜菌體之製備………………….………….……….36
九、篩選反應…….………………….………….……………37
(一)以PEG沉澱重組噬菌體………………………………..37
(二)固體相的篩選…..………………………..………..37
(三)以重組噬菌體再次感染E. coli………………………..39
十、利用ELISA篩選出可與抗原有反應的菌株....……………39
(一)準備可溶性抗體…...………………………………..39
(二)進行ELISA篩選步驟…………………..……………..40
十一、準備重組噬菌體感染E. coli HB2151....……………41
十二、可溶性抗體製備……………………......……………41
(一)製備於supernatant的可溶性抗體…………………….41
(二)製備於perplasmic extract的可溶性抗體……………..42
(三)製備於whole cell extract的可溶性抗體…………….42
十三、確定單鏈抗體蛋白質的表現………......……………42
(一)聚合酶連鎖反應………………………………..….42
(二)酵素切割………….………………………………..43
(三)酵素結合抗體檢定法.……………………………….43
十四、重組質體之構築…………..……......……………43
(一)構築表現抗體基因重組質體………………....43
(二)構築抗體基因轉殖植物重組質體………………....47
(三)黏接反應…………………………………………...50
參、結果………………………………………………………….…51
一、聚合酶連鎖反應產物及gene SOEing結果之電泳分析…..51
二、單鏈抗體基因之定序與譯讀……………..……………..53
三、篩選後所得到的結果……………..……………………..59
四、以酵素結合抗體檢定法分析單鏈抗體的表現..………..59
五、確定單鏈抗體蛋白質的表現………......………………61
(一)聚合酶連鎖反應………………………………..….61
(二)酵素切割………….………………………………..67
(三)酵素結合抗體檢定法.……………………………….70
六、重組質體之構築…………..……......……………….…70
肆、討論…………………………………………….…………………76
伍、參考文獻……………………………………………………………81
陸、附錄…………………………………………………………………93

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