臺灣博碩士論文加值系統

豬生殖與呼吸綜合症（PRRS）是豬全世界經濟最重要的疾病。典型的PRRSV感染豬的免疫功能是遲發性和低水平的中和抗體反應及弱的細胞介導的免疫反應。本病主要引起豬隻早產，流產，死胎，木乃伊胎，重症肺炎，水腫及結膜炎。基因組是長度約15 KB，其中包含9個ORFs。一些其他因素，如動物的年齡和細菌混合感染能夠影響病毒的複製和臨床症狀表現。產氣莢膜梭狀芽胞桿菌是人類和牲畜的主要病原菌。產氣莢膜梭菌腸毒素（CPE）會導致幾種常見的消化道疾病相關的症狀。文獻指出，經由皮下注射免疫小鼠CPE 融合蛋白可活化特異性黏膜免疫反應及全身性黏膜反應。M和GP5蛋白在引發中和性抗體上是必需的，因此我們建構M -GP5 -CPE 融合蛋白，利用原核表現系統表現之，以SDS-APGE及Western blotting 確認產物大小及其抗原性，經皮下注射免疫動物實驗後利用ELISA、細胞激素測定與中和抗體試驗來評估疫苗效力。

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant disease of swine worldwide. Typical immunological features in PRRSV-infected pigs are delayed onset and low level of neutralizing antibodies and a weak cell-mediated immune response. The disease mainly causes premature delivery, miscarriage, stillbirth, mummified fetuses, severe pneumonia, edema and conjunctivitis in pigs. PRRSV belongs to the family Arteriviridae in the order Nidovirales, a family of positive-sense, single stranded linear RNA viruses. The genome is about 15 kb in length which contains nine ORFs. Several other factors such as animal age and bacterial co-infection can influence virus replication and clinical signs. Clostridium perfringens is a major pathogen of humans and livestock. Clostridium perfringens enterotoxin (CPE) causes the symptoms associated with several common gastrointestinal diseases. It's found that subcutaneous immunization of mice with the CPE fusion protein activated antigen-specific mucosal and systemic immune responses. M and GP5 proteins are essential for the neutralizing antibody, so we constructed a M -GP5 -CPE fusion proteins in prokaryotic expression system. The molecular weight and antigenicity were confirmed by SDS-PAGE and Western blot. Then, the subunit vaccine efficacy will be revealed by animal testing with intranasally immunized. Moreover, the immunoassay were tested by ELISA and determination of cytokine and neutralizing antibodies testing.

中文摘要 I
Abstract II
謝誌 IV
目 錄 V
第一章 前言 1
第二章 文獻回顧 3
2.1豬生殖與呼吸綜合症簡介豬生殖與呼吸綜合症 3
（Porcine eproductive and respiratory syndrome ; PRRS) 3
2.1.1豬生殖與呼吸綜合症歷史背景 3
2.1.2豬生殖與呼吸綜合症之命名 3
2.1.3豬生殖與呼吸綜合症病毒之特性 4
2.1.3.1病毒分類 4
2.1.3.2病毒型態與構造 4
2.1.3.3病毒蛋白之功能特性 5
2.1.4豬生殖與呼吸綜合症病毒之臨床症狀 6
2.1.5豬生殖與呼吸綜合症病毒之傳染途徑 7
2.1.6豬生殖與呼吸綜合症病毒之致病機轉 7
2.1.7豬生殖與呼吸綜合症病毒之體液性免疫反應 8
2.1.8豬生殖與呼吸綜合症病毒之細胞性免疫反應 9
2.1.9豬生殖與呼吸綜合症病毒之防治 9
2.2產氣莢膜梭菌腸毒素Clostridium perfringens enterotoxin (CPE) 9
2.2.1產氣莢膜梭菌腸毒素Clostridium perfringens enterotoxin (CPE)之介紹 9
2.2.2 CPE自然感染中的作用 10
2.2.3 CPE之結構與功能 10
2.2.4 CPE受體結合區域 11
2.2.5 CPE毒素之作用機轉 11
2.2.6 緊密結合蛋白（claudins） 11
2.3 研究目的與動機 12
第三章 材料與方法 13
3.1 實驗方法與架構 13
3.2 病材來源 16
3.3實驗一：病毒增殖與定量 16
3.3.1 MARC-145細胞培養 16
3.3.2細胞的凍存 16
3.3.3 PRRS病毒分離與培養 17
3.3.4 PRRS病毒增殖 17
3.3.5 Total RNA萃取 18
3.3.6反轉錄反應 18
3.3.7 Q-PCR PRRS病毒之cDNA 18
3.4實驗二：以原核表現系統建構PRRSV與CPE基因 19
3.4.1 PCR增幅PRRSV基因 19
3.4.2 DNA電泳分析 19
3.4.3 PCR產物回收 19
3.4.5 E coli 勝任細胞的製作 20
3.4.6接合作用（Ligation） 20
3.4.7轉型作用（Transformation） 20
3.4.8藍白篩選 21
3.4.9菌種的保存 21
3.4.10小量質體DNA萃取 21
3.4.11限制酶酵素切割確認重組之質體DNA 22
3.4.12蛋白質誘導 22
3.4.13蛋白質的萃取 22
3.4.14蛋白質電泳分析（SDS-PAGE） 23
3.4.15西方點墨法（Western blot） 24
3.4.16蛋白質純化 25
3.4.17蛋白質的定量 25
3.5實驗三：疫苗免疫動物實驗 26
3.5.1實驗動物組別設定:小鼠組（如附錄） 26
3.5.2實驗動物組別設定:仔豬組（如附錄） 27
3.6免疫效力試驗 27
3.6.1血清抗體檢測 27
3.6.2血清抗體之中和試驗 27
3.6.3血液分離 28
3.6.4細胞激素測定 28
3.6.5 PRRS病毒Copy Number 與病毒定量曲線繪製 29
第四章 結果 30
4.1 PRRSV&;CPE gene 分析 30
4.1.1 PRRSV 專一性引子溫度梯度測試 30
4.1.2 PRRS(GP5+M)-CPE基因合成之Enzyme digestion與PCR確認 30
4.2利用原核表現系統生產PRRS-CPE之重組蛋白 30
4.2.1 IPTG誘導蛋白之SDS-PAGE 30
4.2.2 IPTG誘導蛋白之Western blot 31
4.2.3 PRRS、PRRS-CPE以感染豬血清測其抗原性 31
4.3動物試驗 31
4.3.1小鼠血清抗體測試 31
4.3.2小鼠血清抗體中和試驗 31
4.3.3 小鼠肺泡沖出液（BALF）IgA檢測 32
4.4.1仔豬血清抗體檢測 32
4.4.2仔豬細胞激素測定 32
4.5 PRRSV增殖與分析 32
4.5.1組織檢體定序分型 32
4.5.2 PRRS病毒Q-PCR定量曲線 32
4.5.3以極限稀釋法篩選細胞 33
第五章 討論 49
第六章 參考文獻 52
附錄 65
作者簡介 71

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