造成人體老化的其中一個重要因素便是體細胞所受到的氧化傷害。氧化傷害是經由生理生化反應時釋放出的自由基所造成，因此，抵抗氧化傷害的途徑除了以抗氧化物減少氧化反應外，也可使用自由基的清除者（scanvenger）來減少氧化反應副產物對細胞的傷害。
本研究以未施化學肥料及殺蟲劑之蔬果萃取物（川榮寶源）來評估日常食用之天然蔬果的抗氧化壓力之潛力。寶源乃是將四十種本土性蔬果經製漿除渣後以冷凍乾燥法處理成的粉末狀產品，經水或不同溶劑萃取出可溶性部分，分別以（1）質體DNA模式、（2）蛋白質模式、（3）細胞模式及（4）紅血球模式來評估其在hydroxyl radical的氧化壓力下的保護情形。
以上四種模式分析的結果顯示，寶源水溶性部分可保護氧化壓力對質體DNA及蛋白質的氧化傷害；在細胞的氧化傷害方面其保護力則未觀察到；在紅血球方面，以水溶性部分的抗氧化能力最佳，其餘依序是100% ethanol，ethanol：ethylacetate（1/1；w/w）及50% ethanol。以lysozyme作為比較物質時，在紅血球模式中發現lysozyme對氧化傷害的保護並無幫助。
豬肺炎黴漿菌（Mycoplasma htopneumoniae）是慢性豬流行性肺炎（swin enzootic pneumoniae）的致病原，本菌屬是目前已知最小且能能自我複製的原核生物，但是其致病機制仍不是十分清楚，一般推論，此菌以表面膜蛋白當作吸附分子附著在氣管細胞表面而致病，因此，表面膜蛋白具有發展疫苗的潛力。
本實驗室已將利用免疫篩選法（immunoscreening）自豬肺炎黴漿菌基因庫（M. hyopneumoniae lEMBO3 genomic library）中篩選出的其中一個正反應植珠P42（42 kDa）次選殖至載體pUC18中，本研究則利用PCR的方法將P42的coding sequence自pUC18中複製出來，再選殖到哺乳細胞表現載體pCDNA3中，以此為DNA疫苗，進行免疫反應之評估。
將含有P42的哺乳細胞現載體pCDNA42轉殖到老鼠纖維母細胞NIH3T3中，以RT-PCR的方法可以偵測到P42基因的轉錄，但是以西方墨點法卻觀察不到P42的表現。以pCDNA42免疫老鼠後，觀察老鼠血液中的IgG、IgM，並分析IgG1及IgG2a兩個subclass，其中除了IgM無明顯的上升趨勢外，IgG自第二週起開始上升，第六週達最高點，而IgG1和IgG2a的分析結果顯示兩種subclass的效價沒有明顯的差距。免疫測試如T cell proliferation、CTL response及growth inhibition test等，尚須更進一步的評估。

The oxidative damage to DNA, protein and lipid, may be accumulated and play a role in the process of human cell aging. Oxidative stress may be due to the aerobic respiration and ozone-induced radiation which result in reactive oxygen species known as free radicals. Therefore, the antioxidant and free radical scanvenger which may reduce the oxidative damage, are of great interest, both in academic research and in the business world.
The present study aims to evaluate the antioxidative potential of a very unique vegetable-fruit extract (Tsan-Ron-Bau-Yuan). The extract consists of over forty domestic vegetables and fruits, without using chemical fertilizers and pesticides during their entire growth period, is produced through a sophisticated freeze-dry technology. The anti-hydroxyl radical antioxidative potential were evaluated using the following four methods: (1) the plasmid DNA (2) the protein (3) the cell line and (4) the red blood cell .
The results clearly demonstrat that the aqueous fraction of this extract can remarkably reduce the oxidative damage as evidenced by the DNA and protein model. In addition, the susceptibility of human red cells to oxidative stress can also be alleviated to some extents based on the RBC deformability studies. The efficacy of protection of the oxidative damage mediated by .OH generated by the Fenton’s reaction was in the order of : aquaeous extract >100% ethanol extract > ethanol/ethylacetatee extract > 50% ethanol extract. Conversely, no significant protection to the action of hydrogen peroxide was observed in the cell line. Lysozyme which is ubiquitous among various natural products has negligible contribution as an antioxidant as revealed in the RBC deformability tests.
Swine enzootic pneumoniae （SEP）, is a disease caused by Mycoplasma hyopneumoniae infection, and usually lead to considerable economic loss. Though extensive research during the past years, the molecular mechanism about the infective pathway of M. hyopneumoniae was still elusive. The membrane proteins of this microorganism were considered as critical adhesion molecules and therefore are potential candidates for vaccine development.
Through immunoscreening, we had isolated five recombinant phage clones expressing 10 kDa, 32 kDa, 36 kDa, 42 kDa and 60 kDa antigen proteins from the lEMBL3 library of Mycoplasma hyopneumoniae. The clone carrying the P42 gene was subcloned and further characterized as a heat shock protein gene.
In the present study, the heat-shock protein gene encoding a 42 kDa/ 65 kDa protein（P42/P65）was cloned into the mammalian expression vector pcDNA3 and obtained plasmid pcDNA42. The immune response induced by pcDNA42 was evaluated in mice. The IgG titer was obviously elevated during the first eight weeks with the IgG1 titer slightly higher than IgG2a. However, the IgM titer was not changed signifcantly. Studies on the macrophage activity and T cell cytotoxity were still undergoning.

I、 簡介………………………………………………………………….3
II、研究主題與策略……………………………………………………6
III、材料及方法……………………………………………………….10
IV、結果……………………………………………………………….22
V、討論………………………………………………………………..25
VI、參考資料………………………………………………………….28
VII、圖…………………………………………………………………31
VIII、附圖………………………………………………………………40

II. 對抗豬肺炎黴漿菌之DNA疫苗
英文摘要………………………………………………………………..41
中文摘要………………………………………………………………..42
I、簡介………………………………………………………………….43
II、研究主題與策略…………………………………………………….47
III、材料及方法…………………………………………………………48
IV、結果………………………………………………………………..59
V、討論…………………………………………………………………62
VI、參考資料……………………………………………………………65
VI、圖……………………………………………………………………69

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