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研究生:蔡明安
研究生(外文):Ming-An Tsai
論文名稱:魚類乳酸球菌之分子流行病學、快速診斷及其疫苗效力之研究
論文名稱(外文):Studies on Molecular Epidemiology, Rapid Diagnosis and Vaccine Efficacy of Lactococcus garvieae in Fish
指導教授:陳石柱陳石柱引用關係
指導教授(外文):Shih-Chu Chen
學位類別:博士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:115
中文關鍵詞:乳酸球菌脈衝式膠體電泳分析恆溫圈環型擴增法3-磷酸甘油醛脫氫酶疫苗
外文關鍵詞:Lactococcus garvieaePulsed field gel electrophoresis (PFGE)Loop-mediated isothermal amplification (LAMP)Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)Vaccine
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乳酸球菌(Lactococcus garvieae)是造成世界上多種淡海水魚類乳酸球菌症的重要革蘭氏陽性菌病原,亦是一種在人類與動物間感染病例逐年增加的人畜共通病原。在台灣,本病症常見於夏季,主要感染包括烏魚及淡水長臂大蝦,然而近年來豆仔魚、吳郭魚、石斑魚、日本鰻、赤目豆、黃鰭鯛及牛蛙等養殖水生動物亦發現有感染跡象,因此本研究收集自1999到2006年間台灣76株乳酸球菌菌株、3株日本青魽鰺乳酸球菌菌株及牛源標準株進行其分子流行病學相關性研究,利用限制酶Apa I 與 Sma I切割不同水生動物來源之乳酸球菌染色體DNA後以脈衝式膠體電泳分析(pulsed field gel electrophoresis,PFGE),共獲得10種基因型。而台灣養殖魚類所流行之L. garvieae主要以A1/S1基因型為主(佔96.9%),且具有高度同源性。在毒力試驗結果則以A1/S1及A11a/S11基因型L. garvieae對烏魚及吳郭魚具有較高的致病性。此外亦以乳酸球菌alpha/beta fold family hydrolase 基因設計引子發展恆溫圈環型擴增法(loop-mediated isothermal amplification,LAMP)用於魚類乳酸球菌之檢測,反應溫度及時間所測得之最佳條件分別為60℃與60分鐘,且反應後之產物可利用SYBR Green I直接肉眼判讀。另外本研究設計LAMP引子對於檢測菌落數的靈敏度可高於聚合酶鏈鎖反應100倍,僅300菌落數即可被檢出,在感染魚隻樣本檢測亦優於聚合酶鏈鎖反應。由此結果證明本次建立之LAMP應用在魚類乳酸球菌的檢測是一具特異性、快速、簡易及有效的方法。魚類乳酸球菌在台灣尚未有疫苗成功研發之際,本研究利用大腸桿菌表現系統進行乳酸球菌之3-磷酸甘油醛脫氫酶(glyceraldehyde 3-phosphate dehydrogenase,GAPDH)之選殖並表現出44 kDa的重組GAPDH蛋白,進而分析乳酸球菌之GAPDH其核酸及胺基酸序列發現其他乳酸球菌與相關鏈球菌的相似度高達80.4-100%。另以吳郭魚為測試魚種,分別以乳酸球菌福馬林不活化全菌菌苗、重組GAPDH蛋白(50μg)與兩者組合疫苗進行免疫,對照組則注射生理食鹽水,在免疫後第四週可見單獨全菌菌苗、重組GAPDH蛋白(50μg)與兩者組合疫苗的特異性抗體力價皆有明顯升高,且乳酸球菌全菌與重組GAPDH組合疫苗組之相對存活率達100%,其次分別為全菌菌苗(87.5%)與重組GAPDH蛋白(50%),足見乳酸球菌之重組GAPDH蛋白確實有加乘死菌疫苗之功效。
Lactococcus garvieae is an important gram positive bacterial pathogen that causes lactococcosis in a variety of marine and freshwater fish worldwide. Besides aquatic animals, L. garvieae infection has been observed in cows and human. Therefore, L. garvieae is a zoonotic pathogen of increased clinical significance in animals and humans. In Taiwan, it causes infection grey mullet and giant freshwater prawn during summer. However, L. garvieae infection was also observed in other cultured aquatic animals in Taiwan recently. Thus, it is very important to investigate the relationship between isolates from farmed aquatic animals. The molecular epidemiological relationship among the outbreaks of lactococcosis in grey mullet and other aquatic animals in Taiwan has investigated in this study. Genomic DNA of L. garvieae was digested with restriction endonucleases Apa I and Sma I, and separated by pulsed field gel electrophoresis (PFGE). Ten different L. garvieae genotypes were identified in 74 Taiwanese aquatic animal strains from 1999–2006. Epidemiological analysis of PFGE results indicated that the grey mullet lactococcosis outbreaks in Taiwan was produced by genetically related clones and a high level of genetic homogeneity among the strains in Yunlin, Chiayi and Tainan. In experimental challenges with grey mullet and tilapia, L. garvieae genotypes A1/S1 and A11/S11 showed higher virulence compared to other genotypes. Further, based on use of a loop-mediated isothermal amplification (LAMP) detection protocol, this study attempted to detect L. garvieae in fish by using primer sets that are designed from an alpha/beta fold family hydrolase gene of L. garvieae. Reaction temperatures and time were optimized at 60 ℃for 60 min with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. Additionally, LAMP and conventional polymerase chain reaction (PCR) were similar to each other in terms of the detection limit of DNA. However, detection limit of the LAMP assay was approximately 300 colony forming units (CFU) in pure cultures, 100-fold more sensitive than a PCR. Furthermore, the L. garvieae in spleen of experimentally challenged tilapia were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing specificity, rapid yet simple test for detecting L. garvieae in fish. In Taiwan, commercial vaccine has not been developed and used to prevent L. garvieae infection for fish. In this study, the gene encoding 44 kDa glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of L. garvieae was determined and overexpressed by using the Escherichia coli expression system. Analysis results indicated that sequence analysis GAPDH of L. garvieae nucleotide and amino acid are highly homologous (80.4-100%) to several products of GAPDH from L. garvieae and other Streptococcus-related bacteria. On the other hand, tilapias were immunized intraperitoneally with formalin-killed L. garvieae whole cell, recombinant GAPDH (50μg fish-1) from L. garvieae and both. ISA 763A was used as an adjuvant for vaccine and saline was used as a negative control. Following 4 weeks of immunization, the specific antibody level all vaccine groups significantly increased and GAPDH+WC+ISA exhibited the highest relative percent survival (RPS) at 100 %, followed by fish immunized with WC+ISA or GAPDH+ISA, which had RPS values of 87.5% and 50%, respectively.
目 錄
中文摘要…………………………………..…………………………………Ⅰ
英文摘要………………………………………..……………………………Ⅲ
誌謝……………………………………………………………………………Ⅵ
目錄……………………………………………………………………………Ⅶ
圖表目錄………………………………………………………………………Ⅺ
第1章 緒言……………………………………………………………………1
第2章 文獻回顧………………………………………………………………4
2.1 Lactococcus garvieae病原學……………………………………4
2.1.1命名與分類……………………………………………………………4
2.1.2形態學及生理生化學特性……………………………………………4
2.2 抗原特性及毒力因子……………………………………………………5
2.3致病性及臨床症狀………………………………………………………7
2.4診斷…………………………………………………………………………9
2.4.1生理生化特性鑑定………………………………………………………9
2.4.2聚合酶連鎖反應(polymerase chain reaction,PCR)檢測…10
2.4.3恆溫圈環型擴增法(loop-mediated isothermal amplification, LAMP)……………………………………………………11
2.5流行性病學……………………………………………………………………13
2.5.1表現型鑑定(phenotyping)…………………………………………14
2.5.1.1生物型鑑定(biotyping)…………………………………………14
2.5.1.2血清型鑑定(serotyping)…………………………………………15
2.5.2基因型鑑定….……………………………………………………………15
2.5.3脈衝式電場電泳(pulsed field gel eletrophrosis;PFGE)脈衝 鑑定(pulsotyping…………………………………………………16
2.5.3.1脈衝式電場電泳技術之原理……………………………………………16
2.5.3.2脈衝式電場電泳技術之應用……………………………………………17
2.6疫苗研究………………………………………………………………………19
第3章 材料與方法…………………………………………………………………23
3.1魚類L. garvieae之分子流行病學分析……………………………………23
3.1.1試驗菌株……………………………………………………………………23
3.1.2聚合酶連鎖反應(polymerase chain reaction,PCR)…………23
3.1.2.1 DNA之萃取……………………………………………………………23
3.1.2.2 引子(primer)………………………………………………………24
3.1.2.3 PCR反應條件與程式…………………………………………………24
3.1.3 細菌染色體限制酶分型-脈衝式電泳分析法…………………………27
3.1.3.1 供試驗菌株……………………………………………………………27
3.1.3.2 細菌染色體DNA栓子(plug)之製備………………………………27
3.1.3.3 限制酵素(restriction enzyme)切割及大片段DNA的分離…27
3.1.3.4 電泳分析………………………………………………………………28
3.1.3.5 親緣樹狀圖分析………………………………………………………28
3.1.4 不同基因型L. garvieae對烏魚及吳郭魚之毒力比較………………28
3.2 恆溫式圈環形核酸增幅法(loop-mediated isothermal amplification, LAMP)……………………………………………………29
3.2.1試驗菌株……………………………………………………………………29
3.2.2.1 DNA之萃取……………………………………………………………29
3.2.2.2 alpha/beta fold family hydrolase 基因PCR增幅………29
3.2.3.1 LAMP引子設計…………………………………………………………30
3.2.3.2 PCR反應條件與程式…………………………………………………30
3.2.3.3 LAMP最佳反應條件測試………………………………………………31
3.2.3.4 LAMP特異性試驗………………………………………………………32
3.2.3.5 LAMP與PCR敏感性之比較……………………………………………34
3.2.3.6 LAMP 與 PCR應用於魚類感染組織之檢測評估……………………34
3.3魚類乳酸球菌之疫苗學研究…………………………………………………35
3.3.1 GAPDH重組蛋白表現……………………………………………………35
3.3.1.1引子對的設計……………………………………………………………35
3.3.1.2 GAPDH基因PCR增幅…………………………………………………35
3.3.1.3轉形作用(DH5α)……………………………………………………36
3.3.1.4轉形作用(BL21(DE3))及表現………………………………………36
3.3.1.5蛋白質電泳分析…………………………………………………………37
3.3.1.6重組GAPDH蛋白純化……………………………………………………37
3.3.1.7西方墨漬法(Western blot)………………………………………38
3.3.2吳郭魚La. garvieae重組GAPDH及全菌菌苗免疫試驗………………39
3.3.2.1供試驗菌株………………………………………………………………39
3.3.2.2福馬林不活化全細胞菌苗之製備………………………………………39
3.3.2.3吳郭魚之免疫……………………………………………………………39
3.3.2.4酵素結合免疫吸附法分析(enzyme-linked immunosorbent assay,ELISA)………………………………40
3.3.2.5統計分析…………………………………………………………………40
3.3.2.6重組蛋白保護力試驗……………………………………………………41
第4章 結果…………………………………………………………………………42
4.1魚類乳酸球菌之分子流行病學分析…………………………………………42
4.1.1聚合酶連鎖反應……………………………………………………………42
4.1.2脈衝式電泳分析乳酸球菌之染色體限制酶分型及其親源相關…………42
4.1.3不同基因型L. garvieae對烏魚及吳郭魚之毒力試驗…………………43
4.2恆溫式圈環形核酸增幅法(LAMP)…………………………………………53
4.2.1 alpha/beta fold family hydrolase 基因PCR增幅……………53
4.2.2 LAMP最佳反應條件………………………………………………………53
4.2.3 LAMP特異性試驗……………………………………………………… 53
4.2.4 LAMP敏感性試驗…………………………………………………………61
4.2.5 LAMP與PCR應用於魚類感染組織之檢測評估…………………………61
4.3魚類乳酸球菌之疫苗學研究…………………………………………………66
4.3.1 GAPDH基因PCR增幅……………………………………………………66
4.3.2 GAPDH基因選殖及序列分析……………………………………………66
4.3.3 GAPDH重組蛋白表現、純化及抗原性分析……………………………67
4.3.4免疫保護力試驗……………………………………………………………67
4.3.4.1抗體力價檢測……………………………………………………………67
4.3.4.2保護力試驗…………………………………………………………… 67
第5章 討論…………………………………………………………………………82
參考文獻……………………………………………………………………………91
附錄………………………………………………………………………………109
附錄1 菌種縮寫對照表…………………………………………………………109
附錄2 L. garvieae生理生化性狀……………………………………………110
附錄3 LAMP引子與原理示意圖…………………………………………………111
附錄4 pET151/-TOPO表現載體示意圖………………………………………112
作者簡介…………………………………………………………………………113

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