海洋放線菌已經被公認是具有生物活性且新穎天然物的來源，並廣泛分佈各種海洋環境中。本研究將從高雄港口及墾丁巴士海峽海域之海洋沉積物篩選具抗菌活性的放線菌，以冷凍七天前處理方法及五種選擇性培養基使增加放線菌分離的效率。結果顯示高斯I培養基具最佳放線菌分離效率。總共分離出811株放線菌，在瓊脂碇抗菌試驗中共有70株 (8.63%)對革蘭氏陽性菌 (Staphylococcus aureus、Bacillus subtilis)具有抗菌活性，將生物活性較佳的26株放線菌以16S rDNA進行親緣關係分析，顯示26株放線菌中包括4個屬Streptomyces (21)、Actinoalloteichus (2)、Saccharomonospora (2)及 Nocardiopsis (1)。另外，挑選出抗菌活性較佳的24株放線菌放大培養，利用粗萃物進行細胞毒殺與紙錠擴散試驗，其中分離出KS12-TJ12、BJ01-PL13、BJ01-PL19、BJ01-PL22、BJ02-PL34、BJ03-PL66、BJ02-PL78、BJ01-PLf10、BJ01-PLf31、BJ02-PLf54、BJ03-PLf58、BJ03-PLf59、BJ03-PLf63及BJ03-PLf82共14株，對癌細胞株 (HL-60、K-562與MOLT 4)和6株致病菌擁有較佳的抑制活性，未來將進一步進行天然物的分離、化學結構及相關活性分析，期望這些菌株的活性代謝產物有作為新型藥物開發的潛力。

It is acknowledged that marine actinomycetes have enormous potential as producers of novel natural products with various biological activities. In this study, marine actinomycetes inhabiting marine sediments, collected from Kaohsiung Harbor and Bashi channel, were screened for their abilities in producing secondary metabolites with antimicrobial activity. To increase the abundance of actinomycetes, the sediment samples were pretreated by freezing for seven days. Marine actinomycetes were then enriched and isolated with five different selective media by using agar-based culture techniques. Our results showed that Gause I agar media were the most suitable media for actinomycete isolation. A total of 811 actinomycetes were isolated from six marine sediment samples. 70 isolates (8.63%) showed antimicrobial activity using agar block methods against at least one of six test strains. 26 isolates that displayed high antimicrobial activity were selected for 16S rDNA sequencing. Phylogenetic analysis of the sequences indicated these isolates are members the following four actinomycetal genera: Streptomyces (21), Actinoalloteichus (2), Saccharomonospora (2), and Nocardiopsis (1). Fourteen isolates viz., KS12-TJ12, BJ01-PL13, BJ01-PL19, BJ01-PL22, BJ02-PL34, BJ03-PL66, BJ02-PL78, BJ01-PLf10, BJ01-PLf31, BJ02-PLf54, BJ03-PLf58, BJ03-PLf59, BJ03-PLf63, and BJ03-PLf82, which have high cytotoxicity against cancer cell (HL-60, K-562 and MOLT 4) as well as antimicrobial activity were good candidates for further natural product isolation and characterization of the bioactive components.

圖目錄...............................................VI
表目錄..............................................VII
第一章 緒論.......................................1
1.1前言.............................................1
1.2 微生物與抗生素...................................1
1.3 海洋微生物......................................2
1.4 放線菌概論......................................3
1.5 海洋放線菌......................................4
1.6放線菌的分離及鑑定方法............................5
1.7 研究目的與動機..................................6
第二章 實驗材料與方法...............................11
2.1藥品、儀器與培養基...............................11
2.1.1藥品..........................................11
2.1.2微生物培養基...................................12
2.1.3實驗儀器......................................12
2.1.4生物活性實驗測試菌株...........................13
2.1.5細胞株........................................14
2.2 培養基配置.....................................15
2.2.1選擇性培養基..................................15
2.2.2菌株培養基....................................17
2.3 海底沉積物的採集及放線菌分離培養.................20
2.3.1海底沈積物樣本來源.............................20
2.3.2樣本前處理....................................20
2.3.3放線菌選擇性培養與總菌落數計數.................20
2.4 放線菌之抗菌活性測試與篩選......................21
2.4.1致病菌株培養與測試濃度.........................21
2.4.2抗菌活性初步篩選...............................21
2.4.3 抗菌活性二次篩選..............................22
2.5 海水需求與菌種保存..............................22
2.5.1 海水需求測試..................................23
2.5.2 菌種冷凍保存..................................23
2.6 16s rDNA 序列鑑定放線菌分類.....................23
2.6.1 放線菌基因體DNA萃取與純化.....................23
2.6.2 聚合酵素連鎖反應增幅16s rDNA片段..............24
2.6.3 DNA電泳分析..................................25
2.6.4 凝膠內DNA之純化..............................25
2.6.5 DNA序列分析..................................26
2.6.6 分子親緣關係分析..............................26
2.7 粗萃物抗菌活性與細胞毒殺分析.....................27
2.7.1 放線菌菌液培養與粗萃取........................27
2.7.2 粗萃物之紙錠擴散抗菌試驗......................27
2.7.3 粗萃物之細胞毒殺試驗..........................27
2.7.4 癌細胞株生長抑制試驗..........................28
第三章 結果與討論...................................37
3.1 海洋沈積物之海洋放線菌分離培養...................37
3.2放線菌株抗菌活性與篩選...........................38
3.2.1 抗菌活性初步篩選.............................38
3.2.2抗菌活性二次篩選..............................39
3.3海水需求性測試..................................40
3.4 放線菌株培養液粗萃.............................40
3.5粗萃物之紙錠擴散試驗活性.........................41
3.6 粗萃物之細胞毒殺試驗活性........................42
3.7 16S rDNA序列比對鑑定放線菌......................43
3.8放線菌親緣關係分析...............................43
第四章 結論與建議...................................69
4.1 結論...........................................69
4.1 建議...........................................70
參考文獻...........................................73
 

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