臺灣博碩士論文加值系統

白色念珠菌(Candida albicans)是一種伺機性的二倍體病源真菌，通常共生於人體中，當宿主免疫功能低下或缺損時，白色念珠菌就會引起嚴重感染甚至造成死亡。白色念珠菌具有型態之間轉換的能力，一般有三種型態：酵母菌型、假菌絲型、菌絲型。其致病力與型態轉換能力被認定為有關聯性的。在菌絲生成的調控路徑上，主要是由Cph1p和Efg1p進行調控。實驗證明cph1/cph1 efg1/efg1雙基因剔除株，無法在實驗室條件下生成菌絲型態且在小鼠中失去致病能力。故可由研究白色念珠菌的型態變化能力再進而推測其與致病性的關聯。白色念珠菌可將蛋白質降解成為多肽鏈並吸收含氮的養分以利生長。菌體會分泌天冬胺酸蛋白酶降解蛋白質，再經由特定的胜肽轉運載體吸收至細胞內，其中CaPTR2和 CaPTR22已被研究出是表現胜肽轉運載體的基因，但該PTR家族中的CaPTR3仍屬未知。由於氮的濃度會影響到白色念珠菌的型態變化，故本研究欲測試CaPTR基因失去功能時對型態轉變能力的影響。本研究利用SAT1 flipper cassette構築CaPTR2、CaPTR3和CaPTR22之剔除株及其雙基因組成之剔除株，並測試其性狀。在結果中發現：Captr2和Captr22及其雙基因突變株和野生株並無明顯差異。但是在Captr3及其有關的雙基因突變株中，菌落生成型態及菌落侵犯力和cph1/cph1 efg1/efg1雙基因剔除株較相似。因此CaPTR3對型態變化確實有影響，但詳細的調控路徑以及其與含氮營養源運輸上的角色，仍需要更進一步的研究。

Candida albicans is a commensal diploid fungus found in human. However, it is an opportunistic pathogen. It can cause serious infections, even death, especially in immunocompromised patients. The hyphal formation was regulatd by the transcription factors CaCph1p and CaEfg1p. The cph1/cph1 efg1/efg1 double mutant was defective in filamentous growth, and became avirulent in a mouse model. Therefore, investigating the morphogenesis may infer the virulence of Candida albicans. Candida albicans can use proteins as the sole source of nitrogen for growth. The secretion of aspartic proteinases allows the fungus to digest host proteins to produce peptides for cells to take up via specific transporters. PTR family is known to affect the up-take of peptides. CaPTR2 and CaPTR22 have been verified as peptide transporters, while the function of CaPTR3 is unknown. The concentrations of nitrogen affect the formation of filaments. In this study, I determined the effect of CaPTR null mutation on the morphogenesis of C. albicans, to investigate the connection of virulence and transport of nitrogen source. I used the SAT1 flipper cassette to construct null mutant and double mutant strains of CaPTR2、CaPTR3 and CaPTR22 .Then I determined the effect on the morphogenesis. The results showed that Captr2/Captr2, Captr22/Captr22 and their double mutant strains showed no difference to the wild-type strain. The Captr3/Captr3 null mutant and it’s double mutant strains showed similar phenotypes to cph1/cph1 efg1/efg1 in invasion test and colony morphology. Therefore, CaPTR3 affects the morphogenesis, but the pathway and the roles of CaPTR3 in nitrogen source transporter need to be investigated further.

一、 緒論 1
1.1 白色念珠菌(Candida albicans) 1
1.2 白色念珠菌之型態轉變與致病力之關聯 1
1.3 胜肽轉運載體(Peptide transporter)之介紹 3
1.4 CaPTR2 介紹 3
1.5 CaPTR22 介紹 3
1.6 CaPTR3 介紹 4
1.7 篩選標記之介紹 4
1.8 基因挑選與實驗目的 5
二、 材料與儀器 6
2.1 菌株(Strain) 6
2.1.1 Escherichia coli : DH5α 6
2.1.2 Candida Albicans 6
2.2 質體(Plasmid) 7
2.3 引子 (Primer) 8
2.3.4 篩選用引子 9
2.4 化學藥品 9
2.5 酵素 11
2.6 藥品配製 11
2.6.1 緩衝溶液及溶劑 11
2.6.2 培養基配製 12
2.7 儀器設備 13
三、 方法與步驟 15
3.1大腸桿菌勝任細胞(Competent cell)的製備 15
3.2 質體DNA(plasmid DNA)的萃取 15
3.3 聚合酶連鎖反應(Polymerase Chain Reaction ) 16
3.3.1 Dream TaqTM DNA polymerase 16
3.3.2 TaKaRa Ex TaqTM 16
3.3.3 PCR 溫度控制儀程式設定 17
3.4 洋菜膠電泳(Electrophoresis) 17
3.5 限制酶反應 (Restriction enzyme digestion) 18
3.5.1 製備Cloning 片段 18
3.5.2 質體確認之限制酶反應 18
3.6製備用於 Cloning 片段之純化 18
3.6.1洋菜膠內的 DNA 萃取 (Gel extraction) 18
3.6.2 PCR clean-up kit 19
3.7 接合反應 (Ligation) 19
3.8 大腸桿菌勝任細胞的轉型 (Transformation) 19
3.9 建構 SAT1 flipper cassette 質體以建構白色念珠菌之單套剔除株 (Heterozygote)和雙套剔除株(Homozygote) 20
3.10 白色念珠菌的轉型反應 (Transformation) 20
3.10.1 白色念珠菌勝任細胞之製備 20
3.10.2 電穿孔（Electroporation） 21
3.11 添加 maltose 於培養液中進行 SAT1 flipper cassette 剔除 21
3.12 複製平皿培養法 (Replica plating) 22
3.13 白色念珠菌 Genomic DNA 抽取與純化 22
3.14 南方墨點法 ( Southern blot ) 23
3.14.1 探針 ( Probe ) 的合成 23
3.14.2 轉漬 DNA (Transfer) 23
3.14.3 雜交反應 (Hybridization) 24
3.14.4 免疫偵測 (Detection) 24
3.14.4.1 利用底片成像 24
3.14.4.2利用冷光儀器系統成像 25
3.15 突變株之性狀分析 (Characterization) 25
3.15.1 簡易生物膜定性測驗 (Biofilm formation test) 25
3.15.2 生長曲線 (Growth curve) 25
3.15.3 芽管測試 (Germ tube assay) 26
3.15.4 誘發菌絲生長觀察形態變化 (Colony morphology) 26
3.15.5 侵犯力測試 (Invasion test) 26
四、 結果 27
4.1建構CaPTR2 單套、雙套基因剔除株 27
4.1.1 建構含SAT1 flipper cassette 及CaPTR2 基因之質體pSAT1-PTR2A1B1 27
4.1.2 建構 CaPTR2單套基因剔除菌株(CaPTR2/Captr2)並用PCR進行初步確認 28
4.1.3使用南方墨點法 ( Southern blot ) 檢驗 CaPTR2單套基因剔除株(CaPTR2/Captr2) 28
4.1.4 建構 CaPTR2之雙套基因剔除株並用PCR進行初步確認(Captr2/Captr2) 29
4.1.5 南方墨點法 ( Southern blot ) 檢驗 CaPTR2雙套剔除株(Captr2/Captr2) 29
4.2建構CaPTR3 單套、雙套基因剔除株 30
4.2.1 建構含SAT1 flipper cassette 及CaPTR3 基因之質體pSAT1-PTR3A1B4 30
4.2.3使用南方墨點法 ( Southern blot ) 檢驗 CaPTR3單套基因剔除株(CaPTR3/Captr3) 31
4.2.4 建構 CaPTR3之雙套基因剔除株(Captr3/Captr3)並用PCR進行初步確認 32
4.3.4 建構 CaPTR22之雙套基因剔除株(Captr22/Captr22)並用PCR進行初步確認 35
4.3.5 南方墨點法 ( Southern blot ) 檢驗 CaPTR22雙套剔除株(Captr22/Captr22) 36
4.4建構CaPTR3,CaPTR2雙基因雙套剔除株 36
4.4.1 建構 CaPTR3雙套剔除且CaPTR2單套剔除之菌株(Captr3/Captr3 CaPTR2/Captr2)並用PCR進行初步確認 36
4.4.2 建構 CaPTR3雙套剔除且CaPTR2雙套剔除之雙基因剔除菌株(Captr3/Captr3 Captr2/Captr2)並用PCR進行初步確認 37
4.4.3 以南方墨點法 ( Southern blot ) 檢驗建構 CaPTR3CaPTR2雙基因雙套剔除菌株(Captr3/Captr3 Captr2/Captr2) 37
4.5建構CaPTR3,CaPTR22雙基因雙套剔除株 38
4.5.1 建構 CaPTR3雙套剔除且CaPTR22單套剔除之菌株(Captr3/Captr3 CaPTR22/Captr22)並用PCR進行初步確認 38
4.5.2 以南方墨點法 ( Southern blot ) 檢驗建構 CaPTR3CaPTR22雙套雙基因剔除菌株 39
4.6建構CaPTR2,CaPTR22雙基因雙套剔除株 40
4.6.1 建構 CaPTR2雙套剔除且CaPTR22單套剔除之菌株並用PCR進行初步確認 40
4.6.2建構 CaPTR2雙套剔除且CaPTR22雙套剔除之雙基因剔除菌株(Captr2/Captr2 Captr22/Captr22)並用PCR進行初步確認 41
4.6.3 以南方墨點法 ( Southern blot ) 檢驗建構 CaPTR2CaPTR22雙套剔除(Captr2/Captr2 Captr22/Captr22)之雙基因剔除菌株 41
4.7白色念珠菌CaPTR2、CaPTR3、CaPTR22 各單基因剔除株及雙基因雙套剔除株之性狀分析 42
4.7.1生長曲線測試(Growth Curve) 42
4.7.2芽管生長試驗(Germ tube assay) 43
4.7.3菌落生成型態表面試驗 43
4.7.4侵犯力測試(Germ tube assay) 44
4.7.5簡易生物膜定性實驗 (Biofilm formation test) 45
五、 討論 46
5.1 基因挑選條件 (Biofilm formation test) 46
5.2 以南方墨點法確認CaPTR2剔除株討論 47
5.3以PCR確認pop-out 後 CaPTR3單套基因剔除株 47
5.4以PCR確認CaPTR22雙套基因剔除株時的DNA ladder討論 47
5.5 建構雙套剔除株(double-mutant strain)討論 48
5.6 性狀分析討論 48
5.6.1生長曲線結果探討 48
5.6.2芽管試驗結果探討 48
5.6.3表面皺摺型態觀察結果探討 49
5.6.4侵犯力測試結果探討 49
5.6.4生物膜簡易定性實驗探討 50
5.7結論 50
5.8未來展望 51
六、 參考文獻 52

李克威 (2014) 交大碩士論文。探討剔除白色念珠菌CaHGT6、CaORF19.7566
CaCDR3和CaCDR3CDR4對白色念珠菌型態變化和生長情況之影響
唐子喬 (2015) 交大碩士論文。探討剔除白色念珠菌CaORF19.1105、CaORF19.5285對白色念珠菌型態變化和生長情況之影響
郭郁琳 (2015) 交大碩士論文。探討剔除白色念珠菌 CaORF19.3360、CaORF19.4965 和CaORF19.6268 對白色念珠菌黏附、型態變化和生長情況之影響
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