豬肺炎黴漿菌是導致豬隻慢性肺炎的致病因子，因為肺部病變使得生長速率及飼料換肉率降低，進而造成經濟上的損失。雖然近年來有四種黴漿菌的基因組被定序完成，豬肺炎黴漿菌的基因組定序也接近完成階段，但只有少數基因及其蛋白質被深入研究，而且其分子層次的致病機轉也尚未清楚，分子疫苗之研發也在起步階段。
為了瞭解更多關於抗原決定位結構的資訊，以做為研發對抗此病原菌之分子疫苗的基礎，本研究採用兩個噬菌體呈現逢機七

Mycoplasma hyopneumoniae is the etiologic agent causing chronic pneumonia of swine. The lung lesions of swine produce the slower growth rate and lower feed conversion ratio and finally cause economic loss. Although four genome projects of mycoplasma species had been completed, the genome-sequencing project of M. hyopneumoniae also closed to the finished stage. However, only a few genes and proteins of M. hyopneumoniae have been studied, the molecular pathogenic mechanism remains elusive. The research of molecular vaccine is still preliminary.

In order to obtain more information about epitope structures as the basis to develop molecular vaccine against this pathogen, two phage-displayed random heptapeptides libraries were used to identify epitopes recognized by purified IgG of rabbit anti-M. hyopneumoniae hyperimmune serum in this study. Individual phage clones were isolated and verified the binding specificity to the purified IgG by Western blot analysis and competitive ELISA after three rounds of biopanning. The selected clones were further characterized by DNA sequencing analysis and deduced to amino acid sequences. There are six consensus sequences contained tri- to hepta-peptide existing among the selected phage clones by aligning the sequences of foreign amino acids displaying on pIII protein. The consensus sequences may be serving as crucial epitopes of M. hyopneumoniae. By searching the protein database of M. hyopneumoniae deposited in NCBI, some surface proteins were matched by the selected mimotopes. Like P97, the essential protein for attaching to cilia of swine, the deduced epitopes mainly located at a.a. from 365 to 382, 395 to 403 and 436 to 452, the R1 and R2 repeated sequences also matched by the mimotopes.

To evaluate the potential of these mimotopes as effective vaccine, several phage clones were chosen to immunize mice by intraperitoneal and intranasal administration. There are specific antibody responses to these mimotopes existing in serum IgG, fecal extracts and bronchoalveolar lavage fluids IgA. The serum IgG subclass profiles analysis reveals that these are mainly existed in IgG1 subclass. Base on the results of IgG subclass profiles analysis in sera, the results suggest that the phage-derived vaccines mainly activate Th2 cellular immunity pathway with the strategy used in this study. The similar results were found in the inactivated vaccine. The Th2 activation will promote the elimination of extracellular microorganism. Western blotting analysis showed that each serum raised by the phage clones could recognize 2 to 5 mycoplasma proteins. With the results of growth inhibition assay, we found that the selected phage clones CS4 and 58 are better vaccine candidates and some proteins (97 kDa、56 kDa and 30 kDa) may play crucial roles in block the bacteria growth.

The advantage was taken of the natural property of M13 phage to infect E. coli, which is initiated by the N terminal of pIII coat protein binding with the F pili of E. coli. Plaque reduction tests were proposed to demonstrate the humoral immunity responses induced by phage-derived vaccine containing the antibodies specifically against the foreign peptide displayed on pIII coat protein.

The present results provide more epitope information of M. hyopneumoniae. The mice immunization results reveal that the phage-displayed mimotopes can be used as potential vaccine candidates. The strategy presented in this study can shorten the time course for vaccine development and provide an alternative pathway for searching vaccine candidates against M. hyopneumoniae.

頁次
目錄 …………………………………………………………………………………… Ⅰ
圖次 …………………………………………………………………………………… Ⅲ
表次 …………………………………………………………………………………… Ⅴ
中文摘要 ……………………………………………………………………………… 1
英文摘要 ……………………………………………………………………………… 3
壹、緒言 ……………………………………………………………………………… 5
貳、背景
一、黴漿菌 …………………………………………………………………………… 6
二、豬黴漿菌性肺炎(swine mycoplasma pneumonia) …………………………… 12
三、抗原決定位圖譜分析 …………………………………………………………… 19
四、噬菌體呈現技術 ………………………………………………………………… 19
五、疫苗類型及發展趨勢 …………………………………………………………… 24
六、本實驗室先前的研究成果 ……………………………………………………… 25
七、研究目的與實驗策略 …………………………………………………………… 26
參、材料與方法
一、材料 ……………………………………………………………………………… 27
二、菌種保存雙重玻璃管之開封 …………………………………………………… 29
三、蛋白質含量之測定 ……………………………………………………………… 30
四、兔抗豬肺炎黴漿菌抗體IgG的純化 …………………………………………… 30
五、純化後抗體IgG的活性分析 …………………………………………………… 32
六、噬菌體效價(titer)的測定 …………………………………………………… 32
七、生物親和性篩選 (bio-panning) ……………………………………………… 33
八、接合專一性測試 ………………………………………………………………… 34
九、DNA定序反應 …………………………………………………………………… 35
十、豬肺炎黴漿菌抗原決定位圖譜之分析 ………………………………………… 38
十一、噬菌體抗原的製備 …………………………………………………………… 39
十二、動物免疫 ……………………………………………………………………… 39
十三、抗血清、糞抗體及肺沖洗液的製備 ………………………………………… 40
十四、抗體效價及IgG亞型(subclass)之分析 …………………………………… 42
十五、分析噬菌體免疫產生之抗體對豬肺炎黴漿菌蛋白之辨識及對不同黴漿菌
菌株間的交叉反應 …………………………………………………………… 43
十六、豬肺炎黴漿菌生長抑制測試 ………………………………………………… 44
十七、噬菌斑降減試驗法 …………………………………………………………… 45
肆、結果
一、豬肺炎黴漿菌抗原決定位圖譜分析
1.兔抗豬肺炎黴漿菌抗體IgG的純化及活性分析 ………………………………… 46
2.生物親和性篩選 …………………………………………………………………… 47
3.篩得之噬菌體與純化抗體結合專一性之確認 …………………………………… 47
4.噬菌體外源heptapeptide的基因序列分析 ……………………………………… 48
5.噬菌體呈現外源heptapeptide序列分析 ………………………………………… 48
二、豬肺炎黴漿菌疫苗研發
1.小鼠免疫反應測試 ………………………………………………………………… 50
(1)抗體IgG之效價分析 ……………………………………………………………… 50
(2)劑量效應 ………………………………………………………………………… 50
(3)佐劑效應 ………………………………………………………………………… 51
(4)與市售不活化疫苗(RespiSure)之比較 ………………………………………… 51
(5)血清IgG亞型抗體之分析 ………………………………………………………… 51
(6)糞抗體及肺沖洗液之抗體IgA反應 ……………………………………………… 52
2.篩選之噬菌體誘發的抗體對豬肺炎黴漿菌蛋白之辨識分析 …………………… 52
3.豬肺炎黴漿菌之生長抑制測試 …………………………………………………… 53
4.噬菌斑降減試驗法(plaque reduction test) ………………………………… 54
5.噬菌體疫苗誘發的抗體對不同菌株間之交叉反應測試 ………………………… 55
伍、討論 ……………………………………………………………………………… 56
陸、參考文獻 ………………………………………………………………………… 64
柒、圖表 ……………………………………………………………………………… 74
捌、附錄 ……………………………………………………………………………… 113

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