臺灣博碩士論文加值系統

豬萎縮性鼻炎(Atrophic rhinitis; AR)的主要致病因子為巴氏德桿菌毒素(Pasteurella multocida toxin; PMT)，豬隻一旦感染本病會造成鼻甲介骨萎縮變形，影響全身骨骼發育導致生長遲滯，造成巨大的經濟損失。Bording等(1990)證實PMT類毒素可有效應用於AR，但其產量不高。因此，若能利用重組PMT次單位蛋白進行免疫，則可得到較好的保護效力。在黏膜免疫前置試驗中， PMT次單位疫苗經鼻腔給予已被證實能誘發免疫反應。本實驗欲利用可分泌廣效性殺菌物質-洛德因(reuterin)的洛德乳酸桿菌(Lactobacillus reuteri)作為分泌異源性蛋白質的宿主，並選擇有較佳免疫原性之PMT-Tox1作為免疫之毒素蛋白。將PMT的Tox1片段分別嵌入實驗室先前已構築之p203載體，經比對確認後再將所得之重組質體p203-T1利用電孔轉形至Lactobacillus reuteri DSM20016及野鼠來源之3-3菌株中，以表現PMT-Tox1蛋白。實驗結果顯示p203-T1在Lactobacillus reuteri DSM20016及3-3中均可表現所殖入的PMT-Tox1。進一步的動物實驗更證實所誘發出的IgA ELISA數值與陰性對照組間有顯著差異(P < 0.05)；IgG ELISA 數值則較不明顯。此外，Lactobacillus reuteri 3-3轉形株在腸道的拓殖效果較Lactobacillus reuteri DSM 20016轉形株好；而在接種方式比較上，鼻腔接種較口服接種佳，但無明顯差異。本實驗証實所製備之重組質體疫苗確實可以誘發小鼠的全身性及局部性免疫反應，未來可再提高其抗體力價，以期能應用到豬隻的免疫效力評估。

Atrophic rhinitis (AR), of which the major causative factor has been determined to be the toxin of Pasteurella multocida (PMT), is a widely prevalent, contagious disease of swine, which can cause atrophy of nasal turbinate bones, abnormal skeleton development, and growth retardation, and leading to severe economic losses in the swine industry. Direct application of PMT as vaccine has been proved to be effective in prevention of AR (Boring et al., 1990). However, low yield of PMT from the culture, which was usually regarded as a major difficulty in its vaccine preparation, has prompted scientist to use recombinant PMT as subunit vaccine, of which the effectiveness has also been confirmed via nasal application in animal trials.
The attempt of this study was to employ Lactobacillus reuteri, the producer of a wide-spectrum antimicrobial substance termed reuterin, as a vaccine carrier, bringing PMT into animal’s gastrointestinal tract to induce the mucous membrane-immune response. Hence, the nucleotide sequence of the Tox1 fragment in PMT (PMT-Tox1), being verified to have a strong immune nature, was selected and cloned into the plasmid p203, an expression-secretion vector constructed by our laboratory, to generate the recombinant plasmid p203-T1, which was subsequently transformed into L. reuteri DSM20016 and Lac. reuteri 3-3, respectively.
As expression of the cloned PMT-Tox1 in both L. reuteri strains transformed with p203-T1 (L. reuteri /p203-T1 vaccine) was confirmed to be successful, mice experiment was immediately followed to evaluate the efficacy of our vaccine system. Preliminary results had shown that significant IgA-ELISA titer (P<0.05) was detected in mice applied with L. reuteri /p203-T1, while IgG-ELISA titer in the same experiment was also raised, but not significant. To the effects of different bacteria strains in carrying p203-T1, L. reuteri 3-3/ p203-T1 was found to have a better immune response than that of L. reuteri DSM20016/p203-T1. Also noteworthy was that nasal application in this study generally induce a higher antibody titer than that via the oral administration.
In conclusion, a novel AR vaccine system, which can induce a significant IgA-ELISA titer (P<0.05) against PMT, has been developed in this study. Further swine experiment is needed to evaluate its efficacy in protecting animals from AR infection.

中文摘要.............................................................4
英文摘要.............................................................5
第一章　前言..........................................................7
第二章　文獻探討.......................................................8
第一節、豬萎縮性鼻炎(Atrophic rhinitis；AR)............................8
1、豬萎縮性鼻炎之病因..................................................8
2、巴氏桿菌毒素(P. multocida toxin；PMT)...............................8
2-1、PMT毒素基因......................................................9
2-2、PMT毒素之特性....................................................9
2-3、PMT對動物的影響.................................................10
2-4、PMT影響細胞功能的機制............................................11
3、豬萎縮性鼻炎的致病機制..............................................13
3-1、骨細胞重組(Remodeling)的機制.....................................13
3-2、PMT對造骨細胞的影響..............................................14
3-3、PMT對蝕骨細胞的影響..............................................15
3-4、PMT對細胞素的影響...............................................16
4、PMT的免疫原性.....................................................17
第二節、乳酸菌簡介....................................................17
1、乳酸菌在動物體內之角色..............................................17
2、洛德乳酸桿菌（Lactobacillus reuteri）..............................19
2-1、洛德乳酸桿菌的發現...............................................19
2-2、洛德乳酸桿菌所產生之洛德因(reuterin)..............................19
第三章　材料與方法 ....................................................22
第一節　菌株培養與質體................................................22
一、菌株及質體.......................................................22
二、細菌培養.........................................................22
三、重組質體p203-T1的建立.............................................22
四、E. coli質體的抽取................................................22
五、two-step multiplex PCR..........................................23
六、製備電孔轉形用乳酸菌勝任細胞........................................24
七、電孔轉形.........................................................24
八、L. reuteri質體之抽取.............................................25
第二節 重組質體之表現分析..............................................26
一、重組質體在洛德乳酸桿菌之質體穩定度試驗...............................26
二、洛德乳酸桿菌轉形株蛋白產物之收集....................................26
三、Sodium dodecyl sulfate-polycaryamide gel electrophoresis
（SDS-PAGE）.....................................................27
四、西方墨漬法分析（Western Blot Assay）..............................28
第三節　動物免疫計畫..................................................29
一、免疫計畫.........................................................29
二、洛德乳酸桿菌於小鼠腸道中之拓殖試驗..................................30
三、免疫小鼠肺臟及腸道之免疫蛋白萃取....................................30
四、血清抗體力價（IgG）之偵測..........................................31
五、糞便抗體力價（IgＡ）之偵測.........................................31
六、抗體力價之修正及組間差異之判斷......................................32
第四章　結果.........................................................34
第一節　重組質體p203-T1的構築及鑑定....................................34
第二節　重組質體之功能性分析...........................................34
一、重組質體在洛德乳酸桿菌中的鑑定......................................34
二、質體穩定度測試....................................................34
三、L. reuteri轉形株蛋白產物之收集.....................................35
第三節　動物免疫......................................................35
一、洛德乳酸桿菌於小鼠腸道中之拓殖評估..................................35
二、洛德乳酸桿菌於小鼠腸道及鼻腔中所引起之抗體力價評估....................35
1. IgA (Immunoglobulin A)..........................................35
2. IgG (Immunoglobulin G)..........................................36
第五章　討論.........................................................47
參考文獻............................................................50

圖表目次：

表一、整合之免疫計畫組................................................30
表二、菌株與質體.....................................................33
表三、本實驗所用之引子................................................33

圖一、重組質體p203-T1之構築及其限制酵素圖。.............................37
圖二、膠片電泳圖証實p203-T1的構築。....................................38
圖三、分離自洛德乳酸桿菌之p203-T1重組質體。.............................39
圖四、以PCR技術鑑定洛德乳酸桿菌轉形株之重組質體。........................40
圖五、重組質體p203-T1在L. reuteri DSM 20016及L. reuteri 3-3中之質體穩定度分析。..............................................................41
圖六、以蛋白質電泳(SDS-PAGE)及西方點墨法(Western blot)分析L. reuteri DSM 20016/p203-T1所分泌之蛋白............................................42
圖七、以蛋白質電泳(SDS-PAGE)及西方點墨法(Western blot)分析L. reuteri 3-3/p203-T1所分泌之蛋白。..............................................43
圖八、乳酸菌在小鼠腸道之拓殖研究。......................................44
圖九、洛德乳酸桿菌於小鼠腸道及鼻腔中所引起之IgA抗體力價評估。..............45
圖十、洛德乳酸桿菌於小鼠腸道及鼻腔中所引起之IgG抗體力價評估。..............46

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