臺灣博碩士論文加值系統

　　創傷弧菌是棲息於海水環境中的革蘭氏陰性弧菌，為人類的伺機性致病菌。創傷弧菌感染人體，尤其是具有潛在性疾病的族群，會引發嚴重的傷口感染及敗血症。創傷弧菌會表現許多細胞外的產物，包括莢膜多醣體、蛋白酶、細胞溶解毒素與磷脂酶。我們實驗室之前發現將合成蛋白酶、細胞溶解毒素和磷脂酶的基因都破壞以後，依然不會影響突變株對小鼠的毒力以及對HEp-2細胞的毒殺能力。然而，在我們篩選細胞溶解毒素與磷脂酶雙缺失突變株的過程中，意外的分離到一株不具細胞毒殺能力的雙缺失突變株NY303。NY303不僅喪失了細胞毒殺能力，對小鼠的毒力也明顯的降低。因此，我們推測NY303可能在染色體上某個基因發生了未知的突變，使其喪失了細胞毒殺的能力，而且與細胞毒殺能力相關的蛋白質可能是極為重要的毒力因子。為了瞭解NY303發生未知突變的基因以及進一步發現創傷弧菌可能的毒力因子，在本研究中我們以黏接質體pJRD215為載體構築了創傷弧菌染色體DNA基因庫，接著，將基因庫內的重組質體送入NY303菌體內，再從中篩選出恢復細胞毒殺能力的菌株。我們篩選到一株恢復細胞毒殺能力的菌株JF005。然而，依據定序的結果，此菌株所含的重組質體pJF005並未含有任何與創傷弧菌染色體DNA相似的序列。我們推測pJF005的插入序列可能經由未知的方式嵌入創傷弧菌染色體DNA上而與載體分離。

　　在創傷弧菌所表現的產物中，莢膜多醣體已經被證實是極為重要的毒力因子，而創傷弧菌表現莢膜與否也和其感染小鼠後小鼠的死亡率相關。我們實驗室之前曾以DNA微陣列技術確認創傷弧菌具莢膜的臨床株YJ016染色體序列上與莢膜多醣體合成相關的基因群。我們將一株菌落外觀呈現不透光（具有莢膜）的臨床分離株與2株菌落外觀呈現半透光（不具有莢膜）的環境分離株的染色體DNA，與3個經分析比對後可能與莢膜多醣體合成相關的基因做雜交反應，結果發現雙方只有在開放閱讀架(ORF) VV0361有明顯的差異：不透光的菌株呈現陽性的結果而半透光的菌株則呈現陰性的結果。在本研究中，我們選取了19株環境分離株，以南方墨點法偵測其染色體上是否具有野生株YJ016包含ORF VV0361的基因群內所含的ORFs。我們發現幾乎所有的環境分離株都不含有由ORF VV0341至VV0362之間的ORFs。此外，對人類血清具有抵抗能力的環境株大多含有ORF VV0337至VV0340和ORF VV0363至VV0366間的ORFs，而對人類血清殺菌能力沒有抗性的環境株則大多不具有這些ORFs。為了決定創傷弧菌YJ016在此基因群中的ORFs與莢膜合成的關係，我們將其中的ORF VV0337、ORF VV0364與ORF VV0366分別破壞後，再觀察其菌落形態與對人類血清殺菌力的抗性，結果發現ORF VV0337及ORF VV0364突變株的菌落外觀都變成半透光的狀態，而且也都失去了抵抗人類血清殺菌的能力。相反的， ORF VV0366突變株在菌落外觀與抗血清殺菌能力上與YJ016無明顯差異。ORF VV0337的蛋白質序列與大腸桿菌中參與第一群莢膜多醣體運送到菌體表面的Wza具高度相似性。ORF VV0337下游的DNA序列與其他細菌的基因庫比對後，也發現有許多ORFs與其他參與莢膜多醣體合成的基因有很高的相似度。因此，我們推測從ORF VV0337到ORF VV0365的這一個基因群，可能與第一群莢膜多醣體的表現有關。

　　Vibrio vulnificus is a gram-negative, halophilic, marine bacterium. It causes severe wound infection and fulminant septicemia in humans, particularly in those with underlying diseases. This organism secretes a few extracellular products including capsular polysaccharide (CPS), protease, cytolysin and phospholipase. We have previously found that disruption of all the genes encoding protease, cytolysin and phospholipase did not reduce the bacterial virulence in mice or cytotoxicity to the HEp-2 cells. However, a non-cytotoxic mutant, NY303, was isolated unexpectedly from a phospholipase-cytolysin double-deficient mutant. This non-cytotoxic mutant was shown to be much less virulent in mice, suggesting that cytotoxicity is an important virulence factor. We suspected that this non-cytotoxic mutant might contain an unknown mutation that resulted in loss of cytotoxicity. To identify the mutated gene, we constructed a genomic library of the wild-type strain with a broad-host-range cosmid, pJRD215. We then transferred the recombinant cosmids to NY303, and screened for clones with restored cytotoxicity to the HEp-2 cells. One clone, JF005, was thus identified. The sequence of the insert in the recombinant plasmid, pJF005, isolated from JF005 was determined, and no sequence belonging to V. vulnificus genome was found. We suspect that the insert of pJF005 may have integrated into the chromosome of V. vulnificus by an unknown mechanism.

　　CPS has been shown to be a primary virulence factor of V. vulnificus and correlate with bacterial lethality in mice. To determine the genes in the genome of V. vulnificus YJ016 that are associated with CPS synthesis, we did a comparative genomics with DNA microarray. Three gene clusters annotated as related to CPS synthesis were selected as the probes and hybridized with the target DNAs prepared from one opaque (capsulated) and two translucent (noncapsulated) strains. The results suggested that ORF VV0361 might be associated with capsular synthesis, because it was present in the opaque strain but not in the translucent ones. In this study, we chose 19 environmental strains, including the 2 strains tested with DNA microarray, and surveyed the distribution of the ORFs upstream and downstream of ORFVV0361 (ORF VV0337- ORF VV0367) in these strains by southern blotting analysis. Most of the test strains did not have the ORFs contained in a region from ORF VV0341 to VV0362. In addition, most of the strains that were resistant to the human serum killing effect contained the ORFs located in the regions from ORF VV0337 to VV0340 and ORF VV0363 to VV0366 while most of those that were not resistant to human serum did not. To determine the involvement of genes in this region in CPS biosynthesis of V. vulnificus YJ016, deletions were introduced into ORFs VV0337, VV0364 and VV0366, respectively. ΔVV0337 and ΔVV0364 mutants changed their colonies from opaque to translucent ones and lost the ability to resist human serum killing effect. On the contrary, the ΔVV0366 mutant showed little difference from YJ016 in these two properties. The amino acid sequence of ORF VV0337 is highly homologous to Wza, which is required for transport of the group 1 CPS to the cell surface in Escherichia coli. Some of the ORFs downstream of VV0337 were also found to be highly homologous to the CPS biosynthesis genes in other bacteria. Our data suggest that the ORFs between VV0337 and 0365 maybe associated with the expression of group 1 CPS.

目錄
頁數
壹、中文摘要 i
貳、英文摘要 iii
參、致謝 v
肆、目錄 vii
伍、表目錄 x
陸、圖目錄 xi
柒、符號及縮寫 xii
捌、緒論 1
玖、材料與方法 12
I. 實驗菌株與質體及其保存方法 12
1. 實驗菌株與質體 12
2. 細菌培養液及培養盤的配製 12
3. 菌種保存方法 13
II. 實驗細胞株的保存與培養以及培養液之配製 13
1. 實驗細胞株 13
2. 細胞培養液的配製 13
3. 實驗細胞的次培養方法 14
4. 細胞的保存方法 14
5. 冷凍細胞之解凍 15
6. 細胞計數的方法 15
7. 細胞培養於96孔細胞培養盤的方法 16
III. 核酸技術 16
1. 小量純化DNA的方法 16
2. 商業套件純化質體DNA 17
3. 商業套件純化染色體DNA 18
4. 限制酵素切割DNA 19
5. DNA片段之不完全切割 19
6. DNA電泳 19
7. Pulsed-field gel electrophoresis 20
8. DNA片段之回收 21
9. DNA片段之去磷酸化反應 22
10. DNA接合作用 22
11. 聚合酶連鎖反應 22
12. PCR放大片段之選殖(TA cloning) 23
13. DNA定序 23
IV. 質體轉移方法 23
1. 熱休克轉型作用 23
2. 細菌接合生殖 25
V. 雜交法 25
1. DNA探針製備 25
2. 雜交膜的製備 26
3. 雜交反應 27
VI. 創傷弧菌染色體DNA黏接質體基因庫(cosmid library)之構築 28
VII. 細菌對表皮細胞的毒性試驗 29
VIII. 突變株之分離 29
IX. 互補作用 30
拾、結果 31
I. 創傷弧菌未知毒力基因之探討 31
i. 創傷弧菌黏接質體基因庫(genomic DNA cosmid library)之構築 31
ii. 互補作用(complementation) 31
iii. 篩選回復細胞毒殺能力之含有重組質體的NY303突變株 32
iv. 分析恢復細胞毒殺能力的JF005互補株所含質體上的插入DNA序列 32
II. 參與莢膜多醣體合成與運送的基因群之研究 33
i. 環境分離株株(CG076, CG099 )的基因分佈 33
ii. 表現莢膜與不表現莢膜之環境株抵抗人類血清殺菌能力的測試 34
iii. 比較具有及不具有抵抗人類血清殺菌能力之菌株的基因分佈 35
iv. 創傷弧菌ΔVV0337、ΔVV0364與ΔVV0366突變株之分離 36
v. 創傷弧菌ΔVV0337、ΔVV0364與ΔVV0366突變株之特性分析 37
vi. 創傷弧菌ΔVV0337互補株的篩選及特性分析 38
拾壹、討論 39
拾貳、參考文獻 46
拾參、圖表集 53
拾肆、自述 78

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81. 李寧宜，創傷弧菌磷脂酶之定序及磷脂酶缺失突變株之分離和特性分析。成功大學微生物暨免疫學研究所碩士論文89年6月。