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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16883

Title: 愛德華氏菌溶血毒素口服次單位疫苗可行性及效果之研究
Studies of the availability and the effect of Edwardsiella tarda oral haemolysin subunit vaccine
Authors: Cheng-han Tsai
蔡承翰
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 愛德華氏菌;口服;疫苗;溶血毒素
Edwardsiella tarda;oral;vaccine;haemolysin
Date: 2008
Issue Date: 2011-06-30T08:43:50Z
Abstract: 愛德華氏菌是淡水養殖池中常在水生病源菌, 常造成養殖產業重大損失;目前養殖業者通常會以施用抗生素控制相關疾病,但抗生素的使用容易造成細菌抗藥性,且藥物殘留的問題可能危害食用者的健康;若能開發有效疫苗,便可以預防相關疾病的爆發,也可減少抗生素的施用。口服疫苗是最為方便,對養殖生物的緊迫也最低;但是口服疫苗抗原容易被消化液所破壞,影響其效能,因此有效的保護抗原也是本實驗重點之ㄧ。 本實驗利用愛德華氏菌(haemolysin)基因之專一性探針Ehly與不同菌株(strain)之愛德華氏菌標準菌株進行雜合,所有實驗菌株皆會被Ehly所雜合。將已選殖部份溶血基因之載體pET34b(+)-hlyA從HMS173轉殖至BL21(DE3)大腸桿菌表現系統。以LB培養,IPTG誘導3小時,利用12% SDS-PAGE及西方點漬檢視目標重組蛋白約為37.7 kDa大小。利用His‧Bind Column純化目標蛋白,確認其純化效果,可溶於PBS的目標蛋白佔總蛋白量之0.7478%,內涵體部份目標重組蛋白佔總蛋白量之16.324 %。利用Reagent X及酸性溶液pH 1.0、pH 2.0及pH 3.0及PBS (pH 7.0)對於目標蛋白質進行沉澱,Reagent X濃度在15 mM以下有較佳沉澱效果。在酸沉澱過程中加入蛋白脢K,並不會造成酸沉澱蛋白有任何破壞;再沉澱後,回復其pH值,所有溶出蛋白皆會被蛋白脢K所降解。以胃液處理沉澱蛋白,以可以發現所溶出之蛋白皆會被胃液所破壞;利用10 mM Reagent X (pH1.0)沉澱蛋白,以胃液處理兩小時,剩餘沉澱部分回復亦可以看到目標蛋白的存在。以利用10 mM Reagent X (pH1.0)沉澱150 mg粗萃目標蛋白,以灌食方式投餵吳郭魚,以西方點漬檢視目標蛋白,在60分鐘血清中開始出現目標蛋白,在8小時達到最高量。以注射及投餵方式對吳郭魚施用疫苗,取得魚血抗血清,利用抗血清與愛德華氏菌溶血毒素進行中和反應,注射組分成兩組,實驗魚編號1、2、3為注射1 μg純化蛋白抗原/100 g魚體重,實驗魚編號4、5、6為注射21 μg粗萃蛋白抗原/100 g魚體重;在溶血毒素作用第二個小時後,只剩第四個禮拜實驗魚編號1、3、4、5、6組分別有22、22、21、23、24的中和力價;在投餵組方面,實驗魚編號1、2、3、4、5為投餵2 mg/100 g魚體重,而實驗魚編號6、7、8、9、10為投餵0.2 mg/100 g魚體重;在溶血毒素作用第三個小時後,只剩第五個禮拜的實驗魚編號1、3、4、5、7、9組及第六個禮拜的實驗魚編號1、2、3、5、8組有21的中和力價。
Edwardsiella tarda is a fish pathogen that is commonly distributes in the fresh water of pound. It always cause the serious financial loss since the disease was burst. The farmers of aquaculture usually use antibiotics to control the related disease. But it may course the drug resistance of pathogen when using the antibiotics and the residue of antibiotics may threaten the healthy. It could be prevent the burst of disease and reduce the using of antibiotics if the effective vaccine have been development. The oral vaccine is the most convenient way to use, and it make the lowest stress for aquatic organism. The lower efficacy of orally administered vaccine is the antigen destruction by gastric acid digestion. Protection the antigen from digestion is a point of our experiment. This experiment using the probe Ehly which can specific hybridization with E. tarda hemolysin gene to test the different strain of standard E.tarda. All of the E. tarad we test can be hybridize from Ehly. We transfer the cloning vector pET34b(+)-hlyA from HMS174 to expression systme BL21(DE3). We using the LB broth to incubate the BL21(DE3)/pET34b(+)-hlyA, then using IPTG induction for three hours. The recombinant target protien size is approximately 37.7 kDa when using the 12% SDS-PAGE and western blot to view the lysate protein. We using the His‧Bind Column to purify the target protein, and check the efficiency of purification. The target protein which dissolves in PBS represented 0.7478% of the total protein. And the target protein from inclusion body represented 16.324% of the total protein. We using reagent X and acidic solution pH1.0, pH2.0, pH3.0, PBS(pH7.0) to precipitate the target protein. There is the better precipitation efficiency of Reagent X concentration less than 15 mM. The precipitation will not be digested by proteinase K during the reaction of precipitate. The protein which release from precipitation will be digested by proteinase K after the pH is reversion. We can see that protein which release from precipitation will be digested when treated by gastric acid. The remain precipitation protein which treated the gastric acid after 2 hour have still reveal the target protein. We feed the tilapia by 150 mg lysate target protein which precipitate by 10 mM reagent X (pH1.0), and using the western blot to view the exist of target protein. The target protein was observed from plasma after 1 hour for feeding. There was be highest level of target protein from plasma after 8 hour for feeding. We used the injection and feeding to administrate the hemolysin vaccine, and collect the antiserum. Using the antiserum to neutralize the hemolysin from E. tarda. In the injection group, we divide into two group. The no. 1, 2, 3 are inject 1 μg pure protein antigen per 100 g fish weight, and 4, 5, 6 are inject 21 μg lysate protein antigen per 100 g fish weight. The 1, 3, 7, 8 and 9 fish have22、22、21、23 and 24neutralization titer at the 4’ week after the hemolysin reaction for 2 hour. In the feeding group, we divide into two group. The no. 1, 2, 3, 4, 5 are feed 2 mg lysate protein antigen per 100 g fish weight, and 6, 7, 8, 9, 10 are feed 0.2 mg lysate protein antigen per 100 g fish weight. the 1, 3, 4, 5, 7and 9 fish at the 4’ week and 1, 2, 3, 5 and 8 fish at 5’ week have 21 neutralization titer after the hemolysin.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M94330027
http://ntour.ntou.edu.tw/ir/handle/987654321/16883
Appears in Collections:[水產養殖學系] 博碩士論文

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