豬胸膜肺炎放線桿菌DNA疫苗之免疫特性研究

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(1)國立高雄大學生物科技研究所碩士論文. 猪胸膜肺炎放線桿菌 DNA 疫苗之免疫特性研究. The Immunological Characterization of DNA Vaccine of Actinobacillus pleuropneumoniae. 研究生：蔣仲豪撰指導教授：楊文仁博士. 中華民國九十五年七月.

(2) 謝誌. 兩年的研究生生涯就這樣匆匆的過去，彷彿昨日還在對陌生的環境感到躊躇，今日已要依依不捨的離開熟悉的一切。首先最要感謝的是我的指導教授楊文仁老師如領航者一般，帶領我更深入的體驗分子生物的世界，開闊了我的視野，楊老師對實驗的嚴格要求，訓練了我嚴謹思考、解決問題的能力，為我累積了自信。在這兩年的研究時光中，無論是實驗上或生活上發生了很多令人挫折的事，但楊老師總是充滿關懷的鼓勵著我，陪著我一一克服困難，古人說：「一日為師，終身為父。」雖然楊老師與我的歲數相差不遠，但楊老師對我來說不僅有父親般的溫暖的關懷，亦有朋友般的真誠的鼓勵，誠如西城男孩的歌曲「You Rise Me Up」的歌詞一般：「You raise me up, so I can stand on mountains. You raise me up, to walk on stormy seas. I am strong, when I am on your shoulders. You raise me up, to more than I can be.」因為有楊老師的栽培，才能讓我成為一個實實在在的研究生。此外要感謝於我們實驗室草創時期，在面對實驗物資缺乏的窘境時，慷慨提供我們實驗資源的系主任陳文輝老師、林順富老師、葛孟杰老師、王恆隆老師、施能朗老師以及黃永森老師，由於有各位老師無私的幫助，我的實驗才能順利的完成。另外要感謝我的口試委員林順富老師以及江惠震老師不厭其煩的為我斧正實驗及論文上的缺點，讓我的論文結果更趨完善。還有要感謝我研究所的同學瑋芳、炯中、榕駿、雅楨、睿城、家曄.

(3) 在我研究的路上與我共同打氣，感謝學弟妹炳傑、倩如、錦桐、暐晴、立普對我實驗上的幫忙，以及系辦劉先生與美純於學校公務上的協助，因為有你們的陪伴讓我這兩年的生活更添色彩。最後要感謝我的家人，尤其是我最敬愛的母親藍美麗女士，在我求學的路上給我最大的支持，讓我無後顧之憂的專心研究；在我挫折時給我最大的鼓勵，讓我無時無刻都能有重拾信心，奮發向上的決心。如果沒有我的母親，便沒有今日的我。未來我也將秉持著各位師長的諄諄教誨，以及親友的祝福，滿懷對研究的熱誠向更高的學術殿堂邁進。. 「Where. there. is. a. will,. there. is. a. way」.

(4) 目錄. 頁次目錄 ·············································································································· Ⅰ 表目錄 ·········································································································· Ⅴ 圖目錄 ·········································································································· Ⅵ 中文摘要 ········································································································ 1 英文摘要········································································································ 2 第一章前言 ·································································································· 4 1.1 猪胸膜肺炎放線桿菌 (Actinobacillus pleuropneumoniae) ·············· 4 1.1.1. 猪胸膜肺炎放線桿菌之簡介 ························································ 4. 1.1.2. 猪胸膜肺炎放線桿菌之分類 ························································ 5. 1.1.3. 猪胸膜肺炎放線桿菌之流行病學 ················································ 6. 1.1.4. 猪胸膜肺炎放線桿菌之致病因子 ················································ 6. 1.1.4.1. 纖毛 (fimbriae) ·········································································· 7. 1.1.4.2. 脂多醣 (LPS) ············································································· 7. 1.1.4.3. RTX (repeats-in-toxin)毒素 ······················································· 8. 1.1.4.4. 鐵離子限制因子 (iron-restriction factor) ································· 9. 1.1.5. 目前對猪胸膜肺炎放線桿菌之預防與治療方法 ······················ 10. 1.2 DNA 疫苗 ························································································· 11 1.2.1. DNA 疫苗之發展起源 ································································ 11. 1.2.2. DNA 疫苗之作用機制 ································································ 12. I.

(5) 1.2.3. 配合 DNA 疫苗所使用之佐劑 ···················································· 14. 1.3 猪胸膜肺炎放線桿菌之抗原介紹 ·················································· 15 1.3.1. 文獻上研發對抗猪胸膜肺炎放線桿菌疫苗採用之抗原 ·········· 15. 1.3.2. 本實驗研發 DNA 疫苗所選用之目標抗原 ································ 17. 1.3.2.1. ApxIA ························································································ 19. 1.3.2.2. ApxⅡA ····················································································· 19. 1.3.2.3. OmlA ························································································· 20. 1.3.2.4. HgbA ························································································· 20. 1.4 實驗目的與設計 ·············································································· 21 第二章材料與方法 ···················································································· 22 2.1 猪胸膜肺炎放線桿菌目標抗原基因之選殖 ·································· 22 2.1.1. 猪胸膜肺炎放線桿菌菌株來源 ·················································· 22. 2.1.2. 猪胸膜肺炎放線桿菌菌體培養 ·················································· 22. 2.1.3. 猪胸膜肺炎放線桿菌基因組 DNA 之萃取 ································ 23. 2.1.4. DNA 聚合酶連鎖反應 (polymerase chain reaction, PCR) ········· 24. 2.1.5. 洋菜膠電泳 (agarose gel electrophoresis) ··································· 24. 2.1.6. 目標抗原 DNA 之純化 ································································ 25. 2.2 猪胸膜肺炎放線桿菌目標抗原基因質體之建構 ·························· 25 2.2.1. 以 pcDNATM3.1 表現質體接合目標抗原 DNA ·························· 25. 2.2.2. 質體轉化 (transformation) ·························································· 26. 2.2.3. 質體 DNA 的製備 ········································································ 26. 2.2.4. 質體 DNA 正確性之確認 ···························································· 27. II.

(6) 2.3 猪胸膜肺炎放線桿菌抗原基因質體於活體外 (in vitro)之表現 28 2.3.1. 猪腎細胞 (LLC-PK1)與大腸桿菌 (TOP10)之培養 ················· 28. 2.3.1.1. 猪腎細胞 (LLC-PK1) 之培養 ··············································· 28. 2.3.1.2. 大腸桿菌 (TOP10)之培養 ····················································· 29. 2.3.2. 細胞轉染 (transfection) ······························································· 29. 2.3.3. 硫酸十二酯鈉多醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) ························ 29. 2.3.3.1. 蛋白質樣本製備 ····································································· 29. 2.3.3.2. 製作硫酸十二酯鈉多醯胺凝膠 ············································· 30. 2.3.4. 西方點墨法 (Western blot) ························································· 31. 2.4 DNA 疫苗之動物試驗 ···································································· 32 2.4.1. 實驗動物 ······················································································ 32. 2.4.2. 疫苗免疫策略 ·············································································· 32. 2.4.3. 血清檢體製備 ·············································································· 33. 2.4.4. 酵素連結免疫吸附法 (ELISA) ··················································· 33. 2.4.5. 攻毒試驗 ······················································································ 35. 第三章結果 ································································································ 36 3.1 猪胸膜肺炎放線桿菌菌體培養 ······················································ 36 3.2 猪胸膜肺炎放線桿菌目標抗原基因之選殖 ·································· 36 3.3 猪胸膜肺炎放線桿菌目標抗原基因之建構 ·································· 37 3.4 猪胸膜肺炎放線桿菌抗原基因質體於活體外 (in vitro)之表現 3.4.1. 猪胸膜肺炎放線桿菌抗原基因質體於大腸桿菌 (TOP10). III. 38.

(7) 之表現 ························································································· 38 3.4.2. 猪胸膜肺炎放線桿菌抗原基因質體於猪腎細胞 (LLC-PK1) 之表現 ························································································· 39. 3.5 猪胸膜肺炎放線桿菌 DNA 疫苗所誘發之免疫反應 ···················· 39 3.5.1. 抗體反應 ······················································································ 39. 3.5.2. 猪胸膜肺炎放線桿菌 DNA 疫苗對小鼠抵抗攻毒之保護效果 40. 圖表 ···································································································· 42 第四章討論 ································································································ 66 第五章參考文獻 ························································································ 72. IV.

(8) 表目錄. 頁次表 1 培養基及培養液之配製 ····································································· 42 表 2 緩衝液及實驗試劑之配製 ································································· 43 表 3 PCR 試劑 ···························································································· 45 表 4 合成各基因片段之寡核苷酸引子 ····················································· 46 表 5 四種抗原 DNA 定序結果與 NCBI 基因資料庫比對結果 ················· 47 表 6 各組小鼠血清之抗體效價 ································································· 48. V.

(9) 圖目錄. 頁次圖 1 猪胸膜肺炎放線桿菌各種血清型與 Apx 毒素基因型對照圖 ······· 49 圖 2 猪胸膜肺炎放線桿菌接收鐵離子之路徑 ······································· 50 圖 3 DNA 疫苗之作用機制 ······································································ 51 圖 4 pcDNATM3.1 Directional TOPO vector 基因序列圖譜 ···················· 52 圖 5 猪胸膜肺炎放線桿菌於 BHI plate 上之生長情形 ·························· 53 圖 6 以 PCR 擴增出之 ApxIA、ApxⅡA、OmlA、HgbA 基因片段 ······· 54 圖 7 pcDNAD-ApxIA 質體以限制酶 EcoR V 切割後之基因片段 ········· 55 圖 8 pcDNAD-ApxⅡA 質體與 pcDNA-OmlA 質體以限制酶切割後之基因片段 ······················································································· 56 圖 9 pcDNAD-HgbA 質體以限制酶 EcoR V 切割後之基因片段 ·········· 57 圖 10 pcDNAD-ApxIA 質體定序結果與 NCBI 基因資料庫 ApxIA 基因 (Accession No. AF240779)比對結果 ··································· 58 圖 11 pcDNAD-ApxⅡA 質體定序結果與 NCBI 基因資料庫 ApxⅡA 基因 (Accession No. AF363362)比對結果 ··································· 59 圖 12 pcDNA-OmlA 質體定序結果與 NCBI 基因資料庫 OmlA 基因 (Accession No. AB007572)比對結果 ··································· 60 圖 13 DNA 疫苗轉化過之 E. coli 菌體總蛋白表現之西方點墨法分析 ···· 61 圖 14 DNA 疫苗轉染過之猪腎細胞 (LLC-PK1)總蛋白表現之 SDS-PAGE 分析················································································ 62. VI.

(10) 圖 15 DNA 疫苗轉染過之猪腎細胞 (LLC-PK1)總蛋白表現之西方點墨法分析··············································································· 63 圖 16 ApxIA 組、ApxⅡA 組、OmlA 組與 HgbA 組小鼠於攻毒後不同時間之存活率 ········································································· 64 圖 17 四合一疫苗組、空載體組、市售疫苗組與 PBS 組小鼠於攻毒後不同時間之存活率 ····························································· 65. VII.

(11) 猪胸膜肺炎放線桿菌 DNA 疫苗之免疫特性研究指導教授：楊文仁博士國立高雄大學生物科技研究所學生：蔣仲豪國立高雄大學生物科技研究所. 摘要. 猪胸膜肺炎放線桿菌 (Actinobacillus pleuropneumoniae) 會引起猪隻出血性、纖維性、壞死性胸膜肺炎，造成猪場嚴重之經濟損失。A. pleuropneumoniae 的致病因子包括莢膜多醣、脂多醣、外膜蛋白、吸附因子以及外毒素，其中又以Apx外毒素與毒力最為相關。ApxI、ApxⅡ為A. pleuropneumoniae所分泌之外毒素，ApxI具有很強之溶血性與細胞毒性，ApxⅡ亦具有微弱之溶血性與細胞毒性，前毒素ApxIA與 ApxⅡA分別為ApxI與ApxⅡ的結構蛋白，ApxIA與ApxⅡA分別被ApxIC與ApxⅡC乙醯活化前不具有毒性。外膜蛋白暴露於病原體的表面，被免疫系統辨識而誘發免疫反應的機率較其他非外膜蛋白高，經常被用來作為疫苗研發之標的。此外 A. pleuropneumoniae 要產生完整的毒力需要吸收宿主體內的含鐵蛋白來取得鐵元素，其所能利用的含鐵蛋白包括轉鐵蛋白 (transferrin) 、鐵血紅素 (haem) 、血紅蛋白 (haemoglobin)等。本研究即以ApxIA、ApxⅡA、OmlA (外膜脂蛋白)、HgbA作為目標抗原，研發對抗猪胸膜肺炎放線桿菌之DNA疫苗，將目標抗原的基因選殖入能在哺乳類細胞表現的載體，並以DNA定序分析驗證其正確性，再將此DNA疫苗轉染至猪腎細胞 (LLC-PK1)內，並以西方點墨法檢測蛋白質表現。個別將四種DNA疫苗及包含四種DNA疫苗之四合一疫苗以肌肉注射免疫小鼠，來評估此等DNA疫苗之效用，並以市售之不活化猪胸膜肺炎放線桿菌疫苗作為陽性對照，以空載體、PBS作為陰性對照。除空載體組、PBS組外，第二次免疫後小鼠血清之抗體效價皆有顯著性之增加，免疫過之小鼠於第一次免疫後 24 天以 5x108 colony-forming unit (cfu)第一血清型之猪胸膜肺炎放線桿菌進行攻毒，各組小鼠攻毒後之存活率如下：四合一疫苗組為 67%； ApxIA組為 50%；ApxⅡA組、OmlA組、市售疫苗組為 25%；HgbA組、空載體組、 PBS組為 0%。實驗結果顯示四合一DNA疫苗具有最佳之保護效用，並優於市售疫苗，證實本實驗所研發之DNA疫苗可作為對抗猪胸膜肺炎放線桿菌感染之新策略。關鍵字：猪胸膜肺炎放線桿菌、DNA 疫苗、疫苗、ApxIA、ApxIIA、OmlA、HgbA. 1.

(12) The Immunological Characterization of DNA Vaccine of Actinobacillus pleuropneumoniae Advisor: Dr. Wen-Jen Yang Institute of Biotechnology National University of Kaohsiung Student: Chung-Hao Chiang Institute of Biotechnology National University of Kaohsiung. ABSTRACT Actinobacillus pleuropneumoniae can cause haemorrhagic, fibrinous and necrotic pleuropneumonia in pigs that causes critical economic losses in pig farm. Virulence factors of A. pleuropneumoniae include the capsular polysaccharides, lipopolysaccharides, outer membrane proteins, adhesion factors and exotoxins. Virulence is strongly correlated with the Apx toxins. ApxI and ApxⅡ are exotoxins of A. pleuropneumoniae. ApxI is strongly hemolytic and strongly cytotoxic. ApxⅡ is weakly hemolytic and weakly cytotoxic. Pretoxins ApxIA and ApxⅡA are structural proteins of ApxI and ApxⅡ, respectively. ApxIA and ApxⅡA are non-toxic before they are acetylated by ApxIC and ApxⅡC, respectively. Outer membrane proteins are exposed on the surface of pathogen, which are more accessible for immune system to induce immune responses than other proteins and usually be used as candidates for vaccine development. A. pleuropneumoniae needs to obtain iron from iron-containing proteins of the host to express full virulence. These iron-containing proteins include transferrin, haem, and haemoglobin. In this study, ApxIA, ApxⅡA, OmlA and HgbA were chosen as the target antigens for DNA vaccine development against A. pleuropneumoniae. These genes were cloned to vectors that can express antigens in mammalian system and inserted genes were confirmed by DNA sequencing analysis. To evaluate the potency of these DNA vaccines, each DNA vaccine and a mixture vaccine containing above four DNA constructs (4 in 1 vaccine) were administrated into mice by intramuscular injection. A commercial inactivated A. pleuropneumoniae vaccine also used as positive control, empty vector and PBS as negative control. The titers of antisera increased significantly except empty vector and PBS group after second immunization. These mice were challenged with 5x108 colony-forming unit (cfu) of 2.

(13) serotype 1 of A. pleuropneumoniae after 24 days of first immunization. The survival rate of each group after 5 days of challenge was as following: 67% for 4 in 1 vaccine; 50% for ApxIA vaccine; 25% for ApxIIA, OmlA and commercial inactivated vaccine; 0% for HgbA, empty vector and PBS group. The results showed that the 4 in 1 DNA vaccine could obtain the best protective efficacy and better than the commercial inactivated vaccine. It indicates that these DNA vaccines used in this study could be a new strategy against infection by A. pleuropneumoniae. Keywords: Actinobacillus pleuropneumoniae, DNA vaccine, vaccine, ApxIA, ApxIIA, OmlA, HgbA. 3.

(14) 第一章. 前言. 1.1 猪胸膜肺炎放線桿菌 (Actinobacillus pleuropneumoniae). 1.1.1 猪胸膜肺炎放線桿菌之簡介猪胸膜肺炎放線桿菌的傳播途徑主要是靠空氣與接觸傳染 (Jobert et al., 2000; Lechtenberg et al., 1994; Torremorell et al., 1997)，自然狀態下，猪是主要宿主 (Sebunya et al., 1983)。菌體主要寄生在猪隻之呼吸道中 (Taylor, 1999)，在被感染的猪隻之鼻腔、扁桃腺、中耳腔以及肺部亦可分離出菌體 (Dom et al., 1994; Duff et al., 1996; Sidibe et al., 1993)。各年齡層猪隻皆可能感染猪胸膜肺炎放線桿菌，但以感染 2~3 個月齡之幼猪為主，死亡率高達 20%以上 (Bosse et al., 2002)。根據菌體血清型的不同、猪隻的免疫狀態、能夠到達猪隻肺部的菌體數 …… 等原因，胸膜肺炎 (pleuropneumonia) 可由急性轉變成慢性 (Cruijsen et al., 1995; Hensel et al., 1993; Rogers et al., 1990; Rosendal et al., 1985; Sebunya et al., 1983)。處於急性期的猪隻可能有下列臨床症狀：高燒、呼吸頻率增快、咳嗽、呼吸困難、厭食、運動失調、嘔吐、下痢 (Ajito et al., 1996; Liggett et al., 1987; Rosendal et al., 1985; Taylor, 1999)，口鼻會因為肺部水腫而出現含有血絲的泡沫 (Rosendal et al., 1985; Taylor, 1999)，支氣管常有多型核. 4.

(15) 白血球 (polymorphonuclear leukocyte, PMN) 滲透及纖維沈澱 (fibrin deposition)的現象 (Ajito et al., 1996; Liggett et al., 1987; Rosendal et al., 1985)，還有急性纖維性肺炎、胸膜炎、心包炎、肺部損傷的症狀 (Bosse et al., 2002)。耐受過急性期的猪隻，病情會轉為慢性，在急性期受到損傷的組織會被血纖維蛋白結締組織 (fibrinous connective tissue)所包覆，隔離壞死的組織 (Liggett et al., 1987; Rosendal et al., 1985)。此外，菌體可藏匿在扁桃線的線窩 (crypt)之中，而使猪隻成為帶原者 (Sidibe et al., 1993)。. 1.1.2 猪胸膜肺炎放線桿菌之分類現今 Actinobacillus pleuropneumoniae 被分類為 Pasteurellaceae 科， Actinobacillus 屬，為革蘭氏陰性菌 (gram-negative)。猪胸膜放線桿菌舊稱為 Haemophilus parahemolyticus 或 Haemophilus pleuropneumoniae，屬於嗜血桿菌屬，但根據 DNA 雜交實驗發現其 DNA 和 Actinobacillus lignieresii 較相近，而和 Haemophilus influenzae 相異較大，故將其改歸屬於 Actinobacillus 屬 (Pohl et al., 1983)。根據其生長是否需要由外界攝取菸鹼醯胺腺嘌呤雙核酸 (nicotinamide adenine dinucleotide hydrate, NAD) 可分類成兩種生物型 (biotype) (Taylor, 1999)，第一生物型需要由外界攝取 NAD 才能生長，第二生物型可由特定的 pyridine nucleotides 或其前驅. 5.

(16) 物來自行製造生長所需的 NAD (Niven and Levesque, 1988)。依據菌體莢膜多醣 (capsular polysaccharide)與脂多醣 (lipopolysaccharide)的不同，第一生物型在 1990 年時被分為 12 種血清型 (Perry et al., 1990)，現今已分出 15 種血清型 (Blackall et al., 2002)，第二生物型可分為 6 種血清型 (Schaller et al., 2001)。. 1.1.3 猪胸膜肺炎放線桿菌之流行病學猪放線桿菌胸膜肺炎最早之疑似病例於 1961 年發生在英國，接續於 1963 年在美國及 1964 年在阿根廷發生。目前世界各國皆有此病例發生之報告。台灣地區最早之感染報告為 1976 年，感染的菌株為第一生物型之第五血清型 (徐興鎔等, 1976)，到 1990 年為止，在台灣已有第一生物型的第一血清型 (80.03%)、第二血清型 (3.2%)、第五血清型 (12.6%)、第七血清型 (3.2%)和第八血清型 (0.8%)等血清型存在，其中以第一生物型第一血清型為最主要。. 1.1.4 猪胸膜肺炎放線桿菌之致病因子猪胸膜肺炎放線桿菌之致病因子眾多，包括纖毛 (fimbriae)、脂多醣 (lipopolysaccharide，LPS)、莢膜 (capsule)、RTX (repeats-in-toxin)毒素、鐵離子限制因子 (iron-restriction factor)、IgA 蛋白酶……等，詳細的致病. 6.

(17) 原因目前尚未完全瞭解，藉由致病因子的特性研究可進一步瞭解猪胸膜肺炎放線桿菌致病機轉，並提供研發疫苗的目標抗原選擇之參考。. 1.1.4.1 纖毛 (fimbriae) 纖毛存在於多種病原菌上，其功能與病原菌吸附宿主有關 (Sauer et al., 2000)。目前，第 1、2、7 和第 12 血清型猪胸膜肺炎放線桿菌之纖毛與纖毛次單元 (fimbrial subunit)已被純化出。純化出的纖毛次單元於 N 端之前 12 個胺基酸亦在其他細菌的纖毛次單元上辨識到，且猪胸膜肺炎放線桿菌之纖毛與纖毛次單元皆會與 Moraxella bovis 的第四分泌型纖毛次單元的抗血清有交叉反應 (cross-reaction)的現象 (Zhang et al., 2000)，顯示猪胸膜肺炎放線桿菌與眾多其他菌種在纖毛蛋白的結構上具有保留性。. 1.1.4.2 脂多醣 (LPS) 脂多醣在多種革蘭氏陰性病原菌上扮演了吸附宿主的角色 (Jacques, 1996; Jacques and Paradis, 1998)，脂多醣的存在對猪胸膜肺炎放線桿菌的吸附能力有很大的關連性，且在刺激宿主的免疫系統方面也扮演了很重要的角色。脂多醣為一複合體，主要可分為三個區域：(i) lipid A，固定整個脂多醣於菌體外膜上。 (ii) core oligosaccharide ，包含. 7.

(18) 2-keto-3-deoxyoctulosonic acid (Kdo)與 heptose residues。(iii) O-antigen，包含重複單元的多醣。最初，由於觀察到表現出 smooth type LPS 的菌株比表現出 semi-rough type LPS 的菌株更有吸附能力，故脂多醣在猪胸膜肺炎放線桿菌中被提出可能與吸附有關 (Belanger et al., 1990)。近年來對於脂多醣的研究更加透徹，利用分子生物技術將脂多醣的 outer core 切除之後發現猪胸膜肺炎放線桿菌的吸附能力與毒性大為減弱 (Ramjeet et al., 2005)，由此更確定了脂多醣在猪胸膜肺炎放線桿菌中扮演吸附能力的角色。. 1.1.4.3 RTX (repeats-in-toxin)毒素 RTX 毒素常見於某些革蘭氏陰性菌，RTX 為 repeats-in-toxin 的縮寫，由於此種毒素家族 (family)的特性為具有富含 glycine 的九個胺基酸之. tandem. repeat. 序列. Leu/Ile/Phe-Xaa-Gly-Gly-Xaa-Gly-Asn. /Asp-Asp-Xaa (Xaa 為任意胺基酸) (Rose et al., 1995)。根據猪胸膜肺炎放線桿菌血清型的不同，可能分泌四種 RTX 毒素 (ApxI、ApxⅡ、ApxⅢ、 ApxⅣ)，猪胸膜肺炎放線桿菌主要的毒性以 RTX 為主，即使無菌體的存在，RTX 毒素便能獨立地直接對猪隻造成傷害 (Frey, 1995)。ApxI 具有強烈地溶血性. (Frey and Nicolet, 1988a; Frey and Nicolet, 1988b)，對. phagocytic cell 有強烈地細胞毒性 (Kamp et al., 1991)，第 1 生物型之第. 8.

(19) 1、5、9、10、11 血清型皆會分泌 ApxI (Beck et al., 1994; Frey et al., 1993a; Frey et al., 1993b; Kamp et al., 1994) (圖 1)。ApxⅡ具有輕微的溶血性及細胞毒性，除了第 1 生物型中第 10 血清型外皆會分泌 ApxⅡ (Frey and Nicolet, 1988a; Frey et al., 1992; Kamp et al., 1991)。ApxⅢ不具有溶血性，但對肺胞的巨噬細胞 (macrophage)與嗜中性白血球 (neutrophil)具有強烈地細胞毒性 (Kamp et al., 1991; Rycroft et al., 1991)，第 1 生物型之第 2、3、4、6、8 血清型皆會分泌 ApxⅢ (Kamp et al., 1991; Macdonald and Rycroft, 1992)。ApxⅣ具有輕微地溶血性，第 1 生物型之所有血清型皆會分泌 ApxⅣ，但僅在於感染猪隻後才會分泌，在 in vitro 的狀態下則否(Cho and Chae, 2001; Schaller et al., 1999)。. 1.1.4.4 鐵離子限制因子 (iron-restriction factor) 猪胸膜肺炎放線桿菌需要鐵離子(iron)才能正常地生長與發揮完整的毒性，而猪胸膜肺炎放線桿菌無法吸收猪隻的乳鐵蛋白 (lactoferrin)來當作主要的鐵離子來源 (D'Silva et al., 1995)，因此表現了某些因子 (factor)經由接收含鐵化合物來汲取猪隻身上的鐵離子，以使菌體本身能正常地生長與發揮完整的毒性。譬如，TbpB 蛋白 (又稱為 Tbp2 或 TfbA) 位於外膜上，可接合猪隻的輸鐵蛋白 (transferrin)、血紅素 (haem)及氯高鐵血紅素 (haemin)來汲取鐵離子 (Gerlach et al., 1992; Gonzalez et al.,. 9.

(20) 1995; Wilke et al., 1997)。TbpA 蛋白 (又稱為 Tbp1 或 TfbB)可形成一個穿膜孔道 (transmembrane channel)將 TbpB 蛋白所汲取的鐵離子由膜外運送到內外膜間 (Daban et al., 1996; Gonzalez et al., 1995; Wilke et al., 1997)。此外可接收含鐵化合物的接收器還包括：FhuA 可接收 siderophore (Del Rio et al., 2006; Mikael et al., 2002)，HgbA 可接收血紅蛋白 (haemoglobin) (Srikumar et al., 2004)。. 1.1.5 目前對猪胸膜肺炎放線桿菌之預防與治療方法目前猪放線桿菌胸膜肺炎的防治主要以不活化菌疫苗為主，如安佳藥廠所製售的百齡佳，便是將第 1 生物型之第 1、2、3、4、5、7 血清型菌體不活化後輔以氫氧化鋁佐劑製成疫苗。目前的疫苗往往僅能減少猪隻的死亡率，而無法避免慢性感染。目前以抗生素 Lincomycin 、 Cefamendole、Flumequine、Nitrofurantoin、Cephalocin、Chloramphenicol 等的使用可延遲症狀的發生率與降低猪隻的死亡率，但一旦停藥後，病情將再度復發，無法根除。本病對猪隻的死亡以及飼料換肉率的下降皆對養猪戶造成重大損失，而疫苗的接種以及患病後的治療亦會提高飼養之成本，是故研發出便宜且有效的疫苗便是當務之急。. 10.

(21) 1.2 DNA 疫苗. 1.2.1 DNA 疫苗之發展起源免疫反應於對抗病原的作用上，主要分為體液性免疫反應與細胞性免疫反應，過去的疫苗通常只能引起體液性免疫反應，無法引起細胞性免疫反應來對病原作有效的清除，減毒疫苗雖然能誘發有效的細胞性免疫反應，但其安全性依然令人顧慮。在 1990 年 Wolff 等人首先以報告基因 (reporter gene)注射到肌肉內，發現報告基因能夠在肌肉細胞表現，啟動了 DNA 疫苗研究的序幕 (Wolff et al., 1990)。1993 年 Ulmer 等人證明以 DNA 免疫小鼠可同時產生對抗感冒病毒 (influenza virus)之體液性及細胞性免疫反應 (Ulmer et al., 1993)。此後許多過去在於免疫或治療上困難的疾病也慢慢地以 DNA 疫苗的形式來進行研究，例如：瘧疾、結核病、麻疹、B 型肝炎、C 型肝炎、後天免疫缺乏症候群，乃至於過敏、自體免疫和癌症……等疾病，不只是預防，DNA 疫苗在某些特定的疾病上更可達到治療的效果 (Gurunathan et al., 2000b)，此外，DNA 疫苗可完整地誘發體液性及細胞性免疫反應，目前尚無實驗證據顯示其在生物體內具有危害性。而 DNA 的保存成本較低，無須冷藏運輸鏈的考量。此外， DNA 較蛋白質安定，較不會有變性、變質的風險。在未來，DNA 疫苗的研究及應用勢必會更加的深入及普遍，改善現有傳統疫苗的缺點，提. 11.

(22) 供更佳品質的疫苗。. 1.2.2 DNA 疫苗之作用機制 DNA 疫苗之作用機轉至今仍未十分明朗，目前有三種假說來解釋 DNA 疫苗是如何誘發免疫反應 (Gurunathan et al., 2000a)：第一種假說是將DNA疫苗以肌肉注射入肌肉細胞或以基因槍打入皮膚細胞時，DNA進入肌肉或皮膚細胞內經由轉錄及轉譯作用，製造出抗原蛋白，抗原蛋白進入proteosome中被分解成小片段的peptide，這些小片段的peptide便被送往內質網中與MHC class I分子結合，形成MHC class I-peptide複合體，MHC class I-peptide複合體經過高基氏體再送至細胞膜表面，與Tc cell (cytotoxic T cell)之TCR (T cell receptor)結合，活化Tc cell，進而驅動免疫系統。然而此假說有其破綻，由於肌肉細胞或皮膚細胞與T 細胞接合時，除了MHC class I-peptide複合體與TCR接合外，尚須B7-1 與 B7-2……等分子存在與T細胞之CD28 分子結合才能完全活化T細胞，但肌肉細胞或皮膚細胞並不表現B7-1、B7-2 分子及MHC class II分子，因此＋. 無法活化CD4 TH cell，免疫系統的驅動便受到限制。第二種假說是 DNA 可能直接進入到肌肉組織中的巨噬細胞 (macrophage) 與樹突狀細胞 (dendritic cell) 或皮膚組織中的蘭氏細胞 (langerhan cell)此等抗原呈現細胞 (antigen presenting cell，APC)中，讓抗. 12.

(23) 原呈現細胞表現出抗原蛋白質，進而與MHC class I和MHC class II分子結合，送至細胞表面，活化TH cell，進而驅動免疫系統。第三種假說是DNA進入到肌肉細胞或皮膚細胞中，經轉錄、轉譯作用製造出抗原蛋白，抗原蛋白會主動分泌至細胞外或表現抗原的細胞壞死而釋出抗原蛋白，APC再經由吞噬作用 (phagocytosis)將抗原蛋白吞噬到細胞內，以endosome的形式包覆抗原蛋白，endosome繼而再與溶酶體 (lysosome)結合形成endolysosome，將抗原蛋白分解成小片段的peptide，小片段的抗原peptide再與endosomal lysosome中的MHC class II結合，形成 MHC class II-peptide複合體，之後再被送至細胞表面以接合TH cell，刺激免疫系統的驅動。此外有研究報告顯示，將 DNA 疫苗以肌肉注射的方式免疫老鼠後，在 10 分鐘內將免疫部位的肌肉細胞切除，並不會影響免疫的效果。相對地，將 DNA 疫苗以基因槍 (gene-gun)免疫老鼠的皮膚細胞，在 24 小時內將免疫部位的皮膚細胞切除，結果老鼠的抗體則會降低。這兩項實驗顯示，DNA 疫苗在不同的免疫途徑下，受免疫的組織在免疫初期存在的必要性也不同，可見不同的免疫途徑所誘發出的免疫作用機制也有所不同 (Klinman et al., 1998)。還有研究報告指出，無論是以肌肉注射或基因槍的免疫途徑，皆是由源自骨髓細胞的抗原呈現細胞 (bone marrow-derived APC)呈現出抗原. 13.

(24) 來活化 T 細胞，而非由免疫部位的細胞執行抗原呈現的工作 (Corr et al., 1996; Iwasaki et al., 1997)。此結果間接地否定了第一種假說，因此目前研究者較接受第二或第三種假說。. 1.2.3 配合 DNA 疫苗所使用之佐劑如同其他形式的疫苗一般，DNA 疫苗亦可使用佐劑來增加免疫反應，例如 CpG motif (CpG Oligodeoxynucleotide，CpG ODN)或細胞激素 (cytokine)是常用於 DNA 疫苗中之佐劑。 CpG motif是特定的DNA序列含有未甲基化的 (unmethylated) CG，因為原核生物的基因普遍存在未甲基化的CpG motif，而哺乳類的基因鮮少有CpG motif，即使存在CpG motif也是有甲基化的現象，因此哺乳類之免疫系統會辨識出未甲基化的CpG motif而將之視為原核生物入侵的訊號，活化先天性免疫 (innate immunity)，包括：自然殺手細胞 (NK cell) 與巨噬細胞……等。研究指出將未甲基化的CpG motif插入質體DNA中免疫小鼠可增強毒殺型T細胞 (TC cell)的反應及增加INF-γ的產生，使免疫反應趨向Th1 反應 (Sato et al., 1996)。此外，以含未甲基化的CpG motif 的DNA疫苗與腫瘤抗原一起免疫老鼠之脾臟細胞 (splenocyte)，IFN-γ及 IgG2a的產生亦會增加，顯示CpG motif對蛋白質疫苗亦有增加免疫反應的效用 (Wooldridge et al., 1997)。. 14.

(25) 細胞激素在免疫系統中扮演調節免疫反應的角色，適當地運用不同的細胞激素可增強或抑制免疫反應，將免疫反應導向 Th1 或 Th2 反應路徑。細胞激素 IL-6 (intertleukin-6)由巨噬細胞 (macrophage)、纖維母細胞 (fibroblast)、內皮細胞 (endothelial cell)、T 細胞、B 細胞、角質細胞 (keratinocyte)、血管系膜細胞 (mesangial cell)、膠質細胞 (glial cell)、軟骨細胞 (chondrocyte)及某些腫瘤細胞所分泌 (Van Snick, 1990)，主要的生理功能為刺激 B 細胞產生抗體 (Hirano et al., 1986)；誘發肝臟內急性期蛋白 (acute phase protein)的生成 (Gauldie et al., 1987)；活化 T 細胞 (Tosato and Pike, 1988)；刺激 myeloma、hybridoma 及 plasmacytoma 增生 (Ikebuchi et al., 1987; Ishibashi et al., 1989; Van Damme et al., 1988)。因此以 IL-6 做為疫苗之佐劑可增進免疫反應的作用。. 1.3 猪胸膜肺炎放線桿菌之抗原介紹. 1.3.1 文獻上研發對抗猪胸膜肺炎放線桿菌疫苗採用之抗原猪胸膜肺炎放線桿菌的致病因子包括纖毛 (fimbriae) 、脂多醣 (lipopolysaccharide，LPS)、莢膜 (capsule)、RTX (repeats-in-toxin)毒素、鐵離子限制因子 (iron-restriction factor)、IgA 蛋白酶……等，過去文獻上. 15.

(26) 探討過對抗猪胸膜肺炎放線桿菌之抗原主要分為 RTX 毒素、外膜蛋白、脂多醣、莢膜多醣。 RTX 毒素是很重要的毒力因子 (Beck et al., 1994)，根據猪胸膜肺炎放線桿菌血清型的不同，可能分泌四種 RTX 毒素 (ApxI、ApxⅡ、ApxⅢ、 ApxⅣ)，其中又以 ApxI 擁有最強之溶血性 (Frey and Nicolet, 1988a; Frey and Nicolet, 1988b)及細胞毒性 (Kamp et al., 1991)，研究發現以 ApxI 或 ApxIA 為抗原之重組蛋白疫苗 (Bagdasarian et al., 1999; Seah et al., 2002)；以酵母菌表現 ApxIIA 或同時表現 ApxIA 及 ApxIIA，將酵母菌餵食小鼠，皆能誘發保護性的免疫反應 (Park et al., 2005；Shin et al., 2005)。外膜蛋白位於菌體的最外側，最容易被免疫系統辨識而誘發免疫反應，而鐵離子的攝取是否足夠對猪胸膜肺炎放線桿菌的生長與發揮完整的毒性有相關性，因此位於猪胸膜肺炎放線桿菌外膜上與攝取鐵離子相關的蛋白也被視為研發疫苗的重點。TbpA 與 TbpB 可接合猪隻的 transferrin、haem、haemin 來汲取鐵離子 (Gonzalez et al., 1995; Wilke et al., 1997)，將 tbpA 與 tbpB 兩段基因作缺失 (deletion)所產生之突變菌株感染小鼠，發現小鼠並無產生臨床症狀，並且於無法從小鼠體內分離出突變菌株，表示 TbpA 與 TbpB 的表現與否對猪胸膜肺炎放線桿菌的毒力與生長有很大的相關性 (Baltes et al., 2002)。HgbA (haemoglobin-binding protein)與攝取 heamoglobin 上之鐵離子有關，將 hgbA 基因作缺失. 16.

(27) (deletion)所產生之突變菌株雖可以接合 heamoglobin，但卻無法利用 heamoglobin 上之鐵離子 (Srikumar et al., 2004)。OmlA (outer membrane lipoprotein)位於猪胸膜肺炎放線桿菌之外膜上，以 OmlA 重組蛋白疫苗免疫之猪隻能降低死亡率 (Gerlach et al., 1993)。脂多醣 (LPS) 與猪胸膜肺炎放線桿菌的吸附 (adhesin) 有關 (Belanger et al., 1990)，以去毒性(detoxified) LPS 或 O-polysaccharide- BSA conjugate 免疫猪隻能提高猪隻受感染後 40%的存活率 (Rioux et al., 1998)。莢膜多醣 (capsular polysaccharide)與猪胸膜肺炎放線桿菌的毒性有關，以 capsular polysaccharide-tetanus toxoid conjugate 免疫猪隻能提高猪隻受感染後 60%的存活率 (Andresen et al., 1997)。. 1.3.2 本實驗研發 DNA 疫苗所選用之目標抗原猪胸膜肺炎放線菌最主要之毒力因子為 RTX toxin，過去有研究指出將猪胸膜肺炎放線桿菌之 apxIA 與 apxⅡA 基因作缺失 (deletion)所產生之突變菌株，發現菌體之毒力大為降低 (Boekema et al., 2004)。另外有研究將猪胸膜肺炎放線桿菌之 ApxIA 與 ApxⅡA 蛋白以酵母菌 (Saccharomyces cerevisiae)表達系統表現，將重組之酵母菌餵食小鼠能降低小鼠遭受猪胸膜肺炎放線桿菌感染時之死亡率 (Park et al., 2005；Shin. 17.

(28) et al., 2005)，故本研究選用不具有毒性之 ApxIA 與 ApxⅡA 作為目標抗原，期望能誘發對抗 ApxIA、ApxⅡ之抗體，降低猪胸膜肺炎放線桿菌之毒力，減少猪隻受感染後之損傷與死亡率。製作疫苗常以外膜蛋白作為目標抗原，誘發出的抗體與 Tc 細胞可直接對菌體攻擊，消滅病原。OmlA (outer membrane lipoprotein)位於猪胸膜肺炎放線桿菌之外膜，蛋白質序列在不同的血清型具有很高之同源性，研究發現以 OmlA 免疫後之猪隻遭受猪胸膜肺炎放線桿菌感染時存活率較高 (Gerlach et al., 1993)，故本研究選用 OmlA 作為目標抗原，期望能對抗不同血清型之猪胸膜肺炎放線桿菌，並降低猪隻受感染後之死亡率。猪胸膜肺炎放線桿菌需要鐵離子才能正常地生長與發揮完整的毒性，haemoglobin 是猪胸膜肺炎放線桿菌主要的鐵來源之一，haemoglobin 主要由外膜上之接受器蛋白 HgbA (haemoglobin-binding protein)所接收，進而攝取其中之鐵離子。研究指出，將猪胸膜肺炎放線桿菌之 hgbA 基因作缺失之突變後，菌體雖然依然能接合 haemoglobin，但卻無法利用 haemoglobin 中所含的鐵離子 (Srikumar et al., 2004)。故本研究選用 HgbA 作為目標抗原，期望利用其位於菌體外膜的特性，能誘發細胞免疫反應，直接殺死菌體，並期望能誘發抗體與 HgbA 結合，降低菌體之鐵離子來源，進而降低菌體之毒性。. 18.

(29) 1.3.2.1 ApxIA ApxI 為分子量 105 kD 之外毒素，有強烈地溶血性 (hemolytic activity) (Frey and Nicolet, 1988a; Frey and Nicolet, 1988b)，對 phagocytic cell 有強烈地細胞毒性 (cytotoxic activity) (Kamp et al., 1991)。第 1 生物型之第 1、 5、9、10、11 血清型皆會分泌 ApxI (Beck et al., 1994; Frey et al., 1993a; Frey et al., 1993b; Kamp et al., 1994)。apxI operon 可轉譯出 ApxI，包含四段基因：活化蛋白 (activator)基因 apxIC ，前毒素結構蛋白 (pretoxin structural protein)基因 apxIA，與毒素分泌相關之構造蛋白基因 apxIB 與 apxID (Frey et al., 1994; Gygi et al., 1992)，ApxIA 蛋白 (110 kD)需要 ApxIC 對其進行後修飾作用 (post-translational modification)後才能活化成具有毒性之 ApxI (Gygi et al., 1990)，ApxI 再經過細胞膜上之 ApxIB 與 ApxID 複合體運送至菌體外，形成外毒素的型態。. 1.3.2.2 ApxⅡA ApxⅡ為分子量 103-105 kD 之外毒素，其有輕微的溶血性及細胞毒性，除了第 1 生物型中第 10 血清型外皆會分泌 ApxⅡ (Frey and Nicolet, 1988a; Frey et al., 1992; Kamp et al., 1991)。apxⅡ operon 可轉譯出 ApxⅡ，其包含兩段基因：活化蛋白 (activator)基因 apxⅡC ，前毒素結構(pretoxin structure)蛋白基因 apxⅡA，比較於 apxI operon，apxⅡ operon. 19.