豬肺炎黴漿菌(Mycoplasma hyopneumoniae)引起慢性流行性豬肺炎，此疾病感染率高，造成畜牧業經濟上的損失。豬肺炎黴漿菌是經由飛沫傳染，感染豬隻呼吸道氣管及支氣管黏膜細胞，黏附於細胞表面致病；因目前對於此菌的致病機制並不清楚，且其表面蛋白對於黏膜細胞的附著應有直接關係，故由表面抗原蛋白著手，發展豬肺炎黴漿菌疫苗。
本實驗室初期以免疫篩選法，由l EMBL3 M. hyopneumoniae genomic library挑到五個正反應殖株，其中一個殖株於E coli.中表現出42 kDa (P42)的抗原蛋白質。P42 (1119 bp)為P65 (1803 bp)C端部份，且P42/P65屬於HSP70 family，
本研究即是將Mycoplasma hyopneumoniae表面抗原蛋白P42 (1119 bp, 372 aa)透過E. coli的蛋白質表現系統大量表現及純化。經由電腦軟體PC GENE分析此蛋白質可能的抗原位置，為aa 321~327；接著以hydroxylamine，可專一性的切割蛋白質(Ans-Gly bond)，再以western blotting分析，發現抗原決定位置於近C端的16 kDa片段上，與電腦所預測之抗原決定部位相符合，故選取aa 321 ~ 327共7個胺基酸合成短鏈peptide，並於peptide之N端接上一個carrier protein (diphtheria toxoid, DT)，做為合成peptide分子疫苗。
將純化之蛋白質P42做為重組分子疫苗，分別添加或不加佐劑，並與合成之peptide分子疫苗，三種方式分別免疫動物。經測量Ig titer及細胞素的表現，發現三者皆能很有效的引發實驗動物之免疫反應。在體液性免疫反應方面，實驗發現加入佐劑的疫苗所引發的Ig效價較高，但合成分子疫苗所引發之抗體效價雖高，卻有極大部份是針對carrier protein所產生之抗體，僅少部份是針對P42蛋白質aa 321 ~ 327 peptide所產生；在IgG1/IgG2a比例上來看，添加佐劑會較不加佐劑的實驗動物表現較高的IgG2a；細胞素的表現方面，有加入佐劑的免疫動物會表現細胞素IL-2、IFN-γ及IL-4，而未加入佐劑的免疫動物則只有IL-4表現。以重組分子疫苗及合成peptide分子疫苗所引發之抗體做生長抑制測試，則發現針對重組分子疫苗之抗體能有效的抑制mycoplasma的生長，但針對合成peptide分子疫苗的抗體，則僅有少許抑制生長的功能。
綜合實驗之結果，P42應是一個良好的免疫刺激劑，且針對其所產生的抗體可抑制mycoplasma生長，確實有發展為實用性疫苗之潛力。然而可能因為P42為不完整之HSP70蛋白質(為HSP70之C端)，故無法含有所有HSP70之epitope足以同時引發明顯的humoral以及cellular immune response，僅能較促進其中humoral immune response的活化，未來將可配合添加其它輔佐物質如佐劑、細胞素或T細胞之epitope等，希望有助於對抗豬對於此蛋白質疫苗的發展應較為有利。

Mycoplasma hyopneumoniae causes swine enzootic pneumonia (SEP) and leads to economic loss worldwide. The mechanism of pathogenesis is still not clear. Since this pathogen remains extracellulary after infection, the surface proteins on M. hyopneumoniae should play very important roles in adhering and affecting tracheal mucosal cells. Therefore, the potential of using the surface proteins as the basic to develop molecular vaccine is currently being investigated.
The recombinant clone expressing the 42 kDa protein was isolated from the

目錄

目錄………………………………………………………………… I
表目錄……………………………………………………………… II
圖目錄……………………………………………………………… III
英文摘要(Abstract)………………………………………………… 1
中文摘要…………………………………………………………… 2
緒論………………………………………………………………… 4
研究主題與策略…………………………………………………… 10
材料與方法………………………………………………………… 11
結果………………………………………………………………… 27
討論………………………………………………………………… 33
結論與展望………………………………………………………… 44
參考資料…………………………………………………………… 46
附表………………………………………………………………… 52
附圖………………………………………………………………… 62


表目錄

Table 1 簡稱表……………………………………………………. 52
Table 2 試劑、藥品、酵素及抗體來源…………………………. 54
Table 3 培養液、緩衝液及試劑之配置………………………… 55
Table 4 2X Friis medium………………………………………….. 57
Table 5 Primer for RT-PCR………………………………………. 59
Table 6 重組蛋白質分子疫苗引發之IgG1/IgG2a之比例……… 60
Table 7 Mycoplasma hyopneumonia生長抑制測試…………….. 61


圖目錄

Fig 1 Restriction map and ORF in the 4.4 kb Xmal-HindIII subcloning fragment………………………………………... 62
Fig 2 The 6xHis tagged protein expression vector pQE30……….. 63
Fig 3 The GST fusion protein expression vector pGEX4T-2…….. 64
Fig 4 轉殖株pQE30-P65中6xHis-P65蛋白質表現及純化……. 65
Fig 5 轉殖株pQE30-P42中6xHis-P42蛋白質表現及純化…… 66
Fig 6 轉殖株pGEX4T-P65中GST-P65蛋白質之表現及純化… 67
Fig 7 轉殖株pGEX4T-P42中GST-P42蛋白質之表現及純化… 68
Fig 8 以monospecific anti-P42抗體偵測M. hyopneumonia total cell lysate…………………………………………………… 69
Fig 9a P65蛋白質親水性分析……………………………………. 70
9b P42蛋白質親水性分析……………………………………. 70
Fig 10a Hydroxylamine切割P42切位分析……………………….. 71
10b Hydroxylamine切割P42 之western blotting分析………. 71
Fig 11 重組蛋白質分子疫苗免疫實驗動物之IgG及IgA titer…. 72
Fig 12 合成peptide分子疫苗免疫實驗動物之IgG及IgA titer… 73
Fig 13 以重組蛋白質分子疫苗及合成peptide分子疫苗與其所引發IgG及IgA產生之交叉反應…………………………… 74
Fig 14 重組蛋白質分子疫苗添加或不加佐劑免疫實驗動物之IgG titer……………………………………………………... 75
Fig 15 重組蛋白質分子疫苗添加或不加佐劑免疫實驗動物之IgG1、IgG2a titer…………………………………………… 76
Fig 16 重組蛋白質分子疫苗添加或不加佐劑免疫實驗動物六週後以脾臟細胞total RNA進行RT-PCR…………………… 77
Fig 17 比較轉殖株pGEX4T-P42、pQE30-P42、pQE30-P65所產生之42 kDa蛋白質………………………………………... 78
Fig 18 Mycoplasma hyopneumonia生長抑制測試……………….. 79
Fig 19 Mycoplasma hyopneumoniae與Mycobacterium tuberculosis HSP70胺基酸序列比較……………………… 80

周時羽，國立中山大學生物科學研究所碩士論文
陳瑞貞，國立中山大學生物科學研究所碩士論文
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