臺灣博碩士論文加值系統

霍亂弧菌依其血清型可分為O1-O139種，其中主要能引發霍亂的致病菌為O1及O139；其致病過程中需先經由菌體吸附到腸道細胞，方可增殖，進而釋放出霍亂毒素造成疾病。霍亂弧菌感染人體的第一步驟為吸附作用，且此作用受生長環境的影響。此外，霍亂弧菌雖為海洋菌，但卻可生長在淡水中，不過霍亂弧菌對鹽度的耐受性較弧菌屬的其他細菌低，所以鹽度可能對霍亂弧菌的生理狀況具有一定的影響。因此本論文的目的在探討鹽度對霍亂弧菌吸附的影響，並利用抗體找出與吸附相關的分子。依據霍亂弧菌O139生長曲線，發現對數生長期的O139在室溫下吸附Hela細胞一小時，且細菌與細胞的作用比例為20：1時的吸附力最佳。以不同鹽度的Trypticase Soy Broth (TSB)培養細菌，發現培養於不含鹽及含5%鹽的TSB之細菌其吸附力有明顯下降。然而，經繼代培養後其吸附力並無隨培養代數增加而降低，但若將第十代的菌體接種回0.5%鹽度TSB中，其吸附力有回升的現象。將培養於不同鹽度的O139活菌感染小鼠，結果顯示，在兩倍半致死劑量(LD50)下，培養於0%及5%鹽度中的活菌分別具有50%及40%的致死率，與正控制組相比較分別下降1.8倍及2倍，且小鼠死亡時間亦較正控制組慢。改以口服方式餵食小鼠活菌，發現將菌量提高至1010以上經過72小時觀察後仍未有小鼠死亡。O139生長於0%鹽度及5%鹽度的培養基中，膜蛋白經由膠體分析後，發現有數個大分子量(98kDa~250kDa之間) 的膜蛋白分子與0.5%鹽度之正控制組有所不同。以6-8μg的抗體總量可完全抑制107 個O139對HeLa細胞的吸附作用。若以2μg抗體中和109 個O139後感染小鼠，則有10%的致死率。免疫老鼠進行抑制吸附性分子單株抗體的製備，融合率為40.4%，再以酵素連結免疫測定法(ELISA)進行篩選得到21株對O139有較強反應且生長良好的融合瘤，經過抑制吸附實驗的篩選後，僅得到Y5C1融合瘤產生的抗體可抑制菌體吸附。由上述可知，生長於無鹽或高鹽的環境下，菌體的吸附力及致病力同時有下降趨勢，且小鼠死亡時間亦延後，因此推測鹽度改變造成霍亂弧菌O139致病力的下降可能與吸附能力的下降有關。

The serotypes of Vibrio cholerae have O1-O139.The major virulent Vibrio cholerae are O1 and O139；and the bacteria have to be attachment and colonization of mucosal surface of the intestine , and then secrete cholera toxin (CT) to cause disease.The first step in infection by Vibrio cholerae is adhesion and colonization , and the function is effected by environmental conditions. In addition , although the Vibrio cholerae grow up in the ocean , it also could grow up in fresh water.Because the Vibrio cholerae tolerance of salinity is lower than other bacteria of the Vibrio sp. , so the physiological functions could be effected by the salinity.Depended on the Vibrio cholerae O139 growth curve , we found the O139 would adhere best when the O139 of log phase adhere the Hela cell 1 hr in room temperature and the ratio of bactera to cell is 20 : 1. To incubate the bacteria in different salt concentration Trypticase Soy Broth(TSB) , we found that bacteria incubated in none and 5% NaCl TSB would decreased adhision.However , we found the adhesion is no change to keep on subculture the bacteria in different salt concentration；but the adhesion would be back when the tenth O139 generation be incubated in 0.5% NaCl TSB.To infect the mice with double LD50 O139 which be incubated in different salt concentration , O139 growed in 0% Nacl and 5% Nacl TSB have 50% and 40% death rate , respectively.To Compare with the positive controls , the death rate of them reduced 1.8X and 2X , repectivaly；and the death time was later than the positive controls.Feeding mice with the live bacteria by oral , we find there are no mouse died after 72 hours with bacteria that over 1010 .The membrane proteins of the O139 growed in 0% and 5% Nacl TSB was analyzed by gel , and we found that several membrane protein molecules with high molecular weight (98kDa~250kDa) had difference in positive control(0.5% NaCl).The total qualityof polyclonal antibodies(PAbs) to inhibite the adhesion of 107 bacteria almostly is 6-8μg.To infect mice with 109 O139 which be neutralized by 2μg antibodies , we found O139 could cause the 10% mice dead.Immuning the mice prepare the monoclonal antibody against adhesion factor , and the fusion rate is 40.2%. TSB.Immuned the mouse to prepare the specific MAbs , the fushion rate is 40.4%.To carry out the selection , we get 21 hybridomas which had high positve reaction to O139 with ELISA( enzyme-linked immunosorbent assay).The inhibition of O139 aderence for the second selection , had only one hybridoma YC51. We could know that O139 growed in high NaCl and none NaCl concentration environment would reduce both their adhesion and virelence；and the time of mice dead is increased.Therefore , the adhesion had effection to the virulence of Vibrio cholerae O139.

中文摘要 i
英文摘要 iii
前言 1
文獻回顧 4
材料與方法 12
一、細菌與人類細胞的製備及保存 12
1.細菌的製備及保存 12
2.霍亂弧菌O139之生長曲線 12
3.人類細胞的製備及保存 13
3-1. HeLa細胞 13
3-2. 骨髓瘤細胞 13
二、抗原的製備 14
三、多株抗體的製備 14
1. 小鼠的抗血清 14
2. 小鼠的腹水 15
四、單株抗體的製備 15
1. 動物的免疫 15
2. 融合瘤的製備 16
2-1. 老鼠骨髓瘤細胞(FO)的製備 16
2-2. 老鼠脾臟細胞的製備 16
2-3. 細胞融合 17
2-4. 融合瘤的培養 18
2-5. 融合瘤的篩選 18
2-6. 腹水的製備 19
2-7. 融合瘤的保存 19
五、抗體的純化 20
六、吸附實驗 20
1. 人類HeLa細胞的製備 20
2. 細菌的製備 21
3. 吸附作用的觀察 21
4. 抑制性吸附作用 21
4-1. 小鼠抗血清 21
4-2. 單株抗體 22
七、不同含鹽度對細菌吸附作用的影響 22
1.不同鹽度培養之細菌的吸附實驗 22
2. 繼代培養對細菌吸附能力的影響 23
八、小鼠的感染實驗 23
1. 半致死劑量的測定 23
2. 不同鹽度培養之O139對小鼠之感染 24
3. 抗體保護力的測定 24
4. 霍亂弧菌O139餵食小鼠感染實驗 24
九、吸附因子的分析 25
1. 細菌膜蛋白的抽取 25
2. 蛋白質電泳法 26
2-1. SDS-PAGE 26
2-2. 銀染 26
3. 西方氏免疫點漬法 26
結果 28
一、霍亂弧菌O139的生長與其對HeLa細胞的吸附作用 28
二、不同鹽度對霍亂弧菌O139吸附作用的影響 28
三、培養於不同鹽度下不同繼代的霍亂弧菌O139之吸附作用 29
四、不同鹽度培養之霍亂弧菌O139對小鼠的致死力 29
五、不同鹽度對霍亂弧菌O139膜蛋白組成的影響 30
六、霍亂弧菌之專一性單株抗體 31
七、霍亂弧菌之專一性抗體對O139吸附作用的影響 31
八、霍亂弧菌之專一性抗體對小鼠的保護力 31
討論 32
參考文獻 37
圖 45
表 58
附錄 60
圖表目錄
圖一、實驗策略流程圖 45
圖二、霍亂弧菌O139培養於含鹽度為0.5%的Trypticase soy broth(TSB)中的生長曲線。 46
圖三、霍亂弧菌O139培養於含鹽度為0%的Trypticase soy broth(TSB)中的生長曲線。 47
圖四、霍亂弧菌O139培養於含鹽度為5%的Trypticase soy broth(TSB)中的生長曲線。 48
圖五、霍亂弧菌O139於不同生長時期時的吸附力。 49
圖六、O139菌株與Hela細胞株的吸附作用。 50
圖七、不同生長時期的O139菌株與Hela細胞株的吸附作用。 51
圖八、霍亂弧菌O139於不同鹽度培養後菌體吸附力。 52
圖九、霍亂弧菌O139於不同鹽度繼代培養後的吸附力。 53
圖十、 (A)不同劑量的霍亂弧菌O139活菌感染小鼠後，小鼠存活情形。
(B)取107、108、109及1010個活菌數的小鼠存活率， 畫出回歸曲線已計算半致死劑量(LD50)。 54
圖十一、不同鹽度培養以及經抗體中和後的霍亂弧菌O139感染小鼠後的小鼠存活率。 55
圖十二、培養於不同鹽度下的霍亂弧菌O139之膜蛋白經
SDS-PAGE(12%)膠體分析後的銀染情形。 56
圖十三、霍亂弧菌以不同劑量多株抗體中和作用後的吸附情形。 57
表一、以不同霍亂弧菌菌株作為抗原利用酵素連結免疫測定法(ELISA)
初步篩選獲得的融合瘤細胞株 58
表二、以抗體進行中和作用後利用抑制性吸附法篩選吸附相關的融合瘤
細胞株 59

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