臺灣博碩士論文加值系統

人類介白素7（interleukin 7, IL-7）是細胞激素的一種，是人類T細胞發育和存活所必須。老鼠實驗結果顯示IL-7可改善肺纖維腫、減緩腎癌轉移、降低卵巢癌腫瘤的生長及減緩癌細胞的轉移。故IL-7或許可適用於TCD （T Cell Depleted）病患（如感染HIV、癌症物化療、先天性免疫缺陷、血液疾病等）免疫功能較差之患者作為輔佐療法以增加naive T細胞之新生。本研究目的是希望藉由基因轉殖蕃茄做為生物反應器生產IL-7，並進行細胞生物活性測試以了解此蛋白質的效用。本研究中構築IL7-1和IL7-2藉由農桿菌基因轉殖法將其送入蕃茄。IL7-1含有IL-7及在N端接上一段植物Thaumatin-like protein （TLP）之signal peptide以利分泌到胞外。而IL7-2除了含有TLP之signal peptide外亦於IL-7 N端接上Cholera toxin B subunit（CTB）以探討口服CTB-IL7之功效。經由抗生素篩選和組織培養再生，我們獲得可能之基因轉殖蕃茄共22株，包括含有IL7-1轉殖基因之蕃茄7株以及含有IL7-2轉殖基因之蕃茄15株。PCR分析結果顯示所選取22株再生植株中有7株帶有轉殖基因IL7-1，有15株帶有轉殖基因IL7-2。以南方吸漬法分析時發現22株轉殖株之轉殖基因複本嵌入數皆為1個。以北方吸漬法探測IL-7轉殖基因表現發現所選取之轉殖株IL-7基因皆會表現，然而強弱不一。以西方吸漬法偵測轉殖株之果實發現所選取之4株IL7-1轉殖株只有2株可測得IL-7蛋白質，而IL7-2轉殖株4株皆可測得IL-7蛋白質。從IL7-1和IL7-2轉殖蕃茄果實之萃取液可測得IL-7蛋白質，含量約為1 ~ 54 ng/g果實鮮重。若以一般大小之蕃茄果實重約200克為例，則每顆蕃茄果實可產約0.2

Human interleukin-7 (IL-7) is a cytokine important for human T cell development and survival. Experimental results showed that IL-7 could improve pulmonary fibrosis, retard renal cancer cells metastases, and reduce ovarian carcinoma tumor development and dissemination. In addition, IL-7 can proliferate naive T cells in immunocompromised patients. Therefore, IL-7 has potential for being used as an adjuvant for curing patients with T-cell depletion such as human immunodeficiency virus infection, cancer chemotherapy, congenital lymphodepletion, and hematopoietic stem cell transplantation. The objective of this study was to establish transgenic tomato plants as a bioreactor for producing therapeutic IL-7 proteins. We have successfully introduced the IL7-1 and IL7-2 into tomatoes via Agrobacterium-mediated transformation. IL7-1 contains a signal sequence of the thaumatin-like protein on the N-terminus of the mature IL-7 protein to facilitate secretion of the fusion protein. IL7-2 contains a fusion sequence of the mature cholera toxin B (CTB) subunit and the mature IL-7. CTB serves as a carrier molecule for uptake of biopharmaceutical proteins in the intestine. After selection and regeneration in media with hygromycin and subsequently culturing in soil, a total of 22 putative transgenic tomatoes were obtained, including 7 with the IL7-1 and 15 with the IL7-2. PCR analyses showed that all the putative transgenic tomatoes contain the transgenes. Southern blot analyses revealed that all the transgenic tomatoes have only 1 copy of the transgene. Expression levels of the IL-7 transgenes vary in transgenic tomatoes. We choose the fruits of 8 transgenic tomatoes, 4 with IL7-1 and 4 with IL7-2, for western blot analyses. The results showed that 2 transgenic tomato fruits with IL7-1 expressed the IL-7 protein and all 4 transgenic tomato fruits with IL7-2 expressed the IL-7 protein. The amount of IL-7 proteins in transgenic tomatoes ranged from 1~54 ng/g of fruit. The homozygous T1 transgenic tomatoes were selected for IL-7 bioassay, however, the segregation ratio did not follow Mendelian inheritance.

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
一、研究背景與目的……………………………………………………4
1.1 以植物生產醫藥用蛋白質………………...……………………4
1.1.1以植物生產可食性疫苗………………….......……………4
1.1.2藉由植物生產可食性疫苗之利弊…………………………6
1.2霍亂毒素B次單元（Cholera toxin B subunit, CTB）…..…..…8
1.2.1發展霍亂毒素B次單元之口服醫療用蛋白質……………9
1.3 介白素7 （Interleukin-7, IL-7）…………………..……..…….9
1.3.1 IL-7調節B細胞與T細胞的發育和存活……………..….9
1.3.2 IL-7於醫療之用途…………………...................…...……11
1.3.3人類IL-7的基因結構………………………..……………13
1.4 研究目的…………………………………...……..……………13
二、材料與實驗方法…………………………..…………..……….…..14
2.1植物材料………………………………....…………………...…14
2.2農桿菌品系……………………………..…..……………….…..14
2.3蕃茄基因轉殖…………………………………………....….…..14
2.4植物genomic DNA的抽取…………………..........…………...15
2.5聚合酵素連鎖反應（Polymerase Chain Reaction, PCR）…….15
2.6南方吸漬法（Southern blot analysis）………………….…...…16
2.7蕃茄葉片總RNA的萃取……………………...…….…………18
2.8北方吸漬法（Northern blot analysis）…………………………19
2.9蕃茄蛋白質萃取、透析與定量……………………..………….19
2.10西方吸漬法（Western blot analysis）……….…….......………20
2.11篩選同型結合體（homozygote）……………….……………21
三、結果…………………………………………………………………22
3.1 以農桿菌進行蕃茄基因轉殖……………………………….…22
3.2 以聚合酵素連鎖反應（PCR）偵測蕃茄再生植株轉殖基因…23
3.3 以南方吸漬法分析確認轉殖基因……………….……………23
3.4 以北方吸漬法分析轉殖基因之表現………………….………24
3.5以西方吸漬法分析蛋白質之表現……………………..………25
3.6基因轉殖蕃茄果實之外源基因（IL-7）表現量………………26
四、討論…………………………………………………….…..………27
4.1轉殖基因在各個基因轉殖株之表現不同..………….…………27
4.2基因轉殖蕃茄果實蛋白之表現...................................................27
4.3基因轉殖株之生長狀況……………………………..………….30
4.4篩選同型結合體植株……………………………..…………….30
4.5未來展望.......................................................................................31
五、參考文獻……………………………………………………………34
圖表………………………………………………………..……………43
附錄……………………………………………………………………..56

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