臺灣博碩士論文加值系統

創傷弧菌為海洋性革蘭氏陰性菌，可以在人類和鰻魚造成疾病。只有一部分的創傷弧菌對於鰻魚是有致病力的，而這類的創傷弧菌被歸類為生物二型。這一型的創傷弧菌對鰻魚的致病力是由一普遍存在於此型菌株內的質體所帶來的。過去的研究發現，生物二型毒力質體本身雖然不會散播至其他細菌，但可以在另一存在於大多數生物二型菌株內之接合質體的幫助下，在此型菌株間以約10-2的頻率傳送。在本研究中，我探討了生物二型毒力質體pR99由一株生物二型野生株CECT4999轉移至三株生物一型臨床株SW058、SW059、YJ016和三株生物一型環境株CG024、CG027、CG028的頻率。為了方便篩選出帶有毒力質體pR99、接合質體pC4602-1，或兩者兼具之接合株(transconjugants)，我們將氯黴素抗藥基因cat和安比西林(ampicillin)抗藥(Apr)基因分別插入pR99和pC4602-1中。結果顯示，pR99::cat確實可以由生物二型菌株傳送至生物一型環境株和臨床中。pR99::cat或者是pR99::cat和pC4602-1::Apr兩者同時轉移的頻率在CG024、CG028、SW059約為10-3，在另三株則約為10-5~10-6。將帶有pC4602-1::Apr之YJ016接合菌株和YJ016進行接合作用後，發現該質體之轉移頻率並沒有提高，排除了限制修飾系統(restriction modification)阻礙pC4602-1::Apr轉移的可能性。此外，和生物二型野生株CECT4999相比，帶有pR99::cat之接合株對鰻魚血清的殺菌作用只有部分的抗性。進一步觀察pR99質體套數和此質體上一傳遞對鰻魚血清的抗性之基因vep07 的mRNA的表現量，發現均與CECT4999相似。然而，部份接合株的外膜上Vep07的表現量較CECT4999低許多，其它的接合株則是和CECT4999相當。此外，將生物一型接合株HC131和HC147的vep07刪除，並不影響此兩菌株對鰻魚血清中的抗性。由此推測，Vep07可能與生物一型接合株對鰻魚血清的抗性無關。

Vibrio vulnificus, a gram-negative estuarine bacterial species, is pathogenic to humans and eels. Only a fraction of V. vulnificus strains, classified as biotype (BT) 2, is virulent for the eel, and the virulence is conferred by a common plasmid in the BT2 strains. It has been shown previously that the BT2 virulence plasmid, though itself is not transmissible, could be transferred between the BT2 strains with a frequency of about 10-2 in the presence of a conjugative plasmid found in most of BT2 strains. In this study, we investigated the transmissibility of a BT2 virulence plasmid, pR99 possessed by strain CECT4999, to three clinical BT1 strains, SW058, SW059 and YJ016, and three environmental BT1 isolates, CG024, CG027 and CG028. The plasmids pR99 and pC4602-1, a BT2 conjugative plasmid, were inserted with chloramphenicol- and ampicillin-resistance cassettes, respectively, for the selection of transconjugants with either or both plasmids. The results indicate that pR99 could be transferred from a BT2 strain into both environmental and clinical BT1 isolates. The frequency of transferring pR99 alone or both pR99 and pC4602-1 were about 10-5~10-6 except for CG024, CG028 and SW059, which showed a frequency of about 10-3. The role of restriction modification systems in preventing uptake of the BT2 plasmid pC4602-1 by the BT1 strains has been excluded as the conjugation frequency of transferring this plasmid from the transconjugant of strain YJ016 back to YJ016 was not elevated. Compared to the BT2 strain, CECT4999, the pR99-possessing BT1 derivatives were only partially resistant to eel serum killing effect. The copy number of pR99 and the mRNA level of vep07, a gene in pR99 that confers the resistance to eel serum, in the BT1 transconjugants were similar to those in strain CECT4999. However, some transconjugants expressed much less Vep07 than strain CECT4999 on the outer membrane while the others expressed similar amount of Vep07 to that of CECT4999. In addition, deletion of vep07 in BT1 transconjugants HC131 and HC147 did not affect the resistance of these two strains to eel serum. These results suggest that Vep07 may not be associated with the resistance of BT1 transconjugants to eel serum killing effect.

中文摘要i
Abstractii
誌謝iii
目錄v
表目錄vii
圖目錄viii
符號及縮寫ix
緒論1
創傷弧菌(Vibrio vulnificus) 1
生物型(biotypes) 1
生物一型(biotype 1) 1
生物二型(biotype 2) 2
生物三型(biotype 3) 3
創傷弧菌致病機轉 4
材料與方法 8
I、 實驗菌株、質體及核酸引子 8
1.實驗菌株 8
2.實驗菌種培養及保存方法 8
3. 質體 8
4. 核酸引子 8
II、 實驗方法 9
? 基礎核酸技術 9
(1) 商業化套件萃取DNA 9
(2) 小量純化質體DNA 9
(3) 創傷弧菌染色體DNA之萃取 10
(4) 商業套件純化染色體DNA 10
(5) 聚合?連鎖反應 (PCR) 11
(6) PCR放大片段之選殖 (TA cloning) 11
(7) 限制?切割DNA 11
(8) DNA電泳分析 12
(9) DNA片段之分離回收 12
(10) DNA片段之去磷酸化反應 12
(11) DNA片段之黏端補齊反應 13
(12) DNA接合反應 13
(13) 以人類血清誘導細菌基因之表現 14
(14) 細菌RNA之萃取 14
(15) 細菌mRNA反轉錄作用及聚合?連鎖反應(RT-PCR) 15
III、 創傷弧菌突變株之製備 15
(1) 創傷弧菌突變株之製備 15
(2) 鏈黴素(streptomycin)自然突變株之製備 16
IV、 DNA序列決定 17
V、 細菌生長曲線之測定 17
VI、 細菌對鰻魚血清抗性試驗 17
VII、 細菌質體套數分析方法 18
VIII、 蛋白質分析方法 18
? 蛋白質的製備 18
(1) 細菌蛋白質之誘導 18
(2) 全細胞非溶解性蛋白質 (total insoluble proteins) 19
(3) 外膜蛋白質 (membrane proteins) 19
(4) 蛋白質樣品之定量 20
? 蛋白質電泳分析 20
(1) 硫酸十二酯鈉聚丙烯醯胺膠體電泳 (SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 20
(2) 蛋白質染色 (Coomassie blue stain) 21
(3) 西方墨點法 (Western-blot) 21
結果22
I. 創傷弧菌生物二型質體轉移頻率之分析 22
II. 接合株的特性分析 24
II-1. 接合株對鰻魚血清的抗性 24
II-2. 生物二型毒力質體上的毒力基因在接合株中的表現量 25
II-3. 生物二型毒力質體在接合株中的套數 26
II-4. 生物一型接合株之vep07突變株對鰻魚血清之抗性 26
III. 限制修飾系統(restriction-modification systems)在質體轉移中所扮演的角色 27
III-1. 創傷弧菌生物一型菌株YJ016與生物二型菌株CECT4999之限制修飾系統的生物資學分析 27
III-2. 限制修飾系統在各個創傷弧菌生物一型菌株之分析 28
III-3. 限制修飾系統在生物二型質體pC4602-1::Apr由生物二型轉移至生物一型中之角色 28
討論29
參考文獻 34
自述69

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