臺灣博碩士論文加值系統

豬生殖與呼吸綜合症 (porcine reproductive and respiratory syndrome; PRRS) 為豬隻常見疾病之ㄧ，可造成母豬繁殖障礙及仔豬間質性肺炎。PRRS病毒 (PRRSV) 屬於Arteriviridae的正股線狀RNA病毒，基因大小約15 kb，包含8個開放性閱讀區(open reading frames; ORF)，其中ORF5負責轉譯26-30 kDa的醣化封套蛋白 (GP5蛋白)。本研究的目的是以重組GP5蛋白製作 PRRSV 檢測試劑。PRRSV先增殖並以RT-PCR方式增幅OPF5片段，再將此ORF5片段裝載入pET 32a 載體以大腸桿菌表現系統表現此重組蛋白，藉由鎳離子純化管柱純化蛋白，再利用抗his抗體及被PRRSV感染之豬隻血清進行西方點墨法和ELISA，以確認GP5蛋白抗原性。另外應用56隻母豬血清檢體，以GP5製作之檢測試劑與商業化Bio Chek PRRSV ELISA Kit比較，結果顯示GP5所開發之檢測試劑具有高度特異性 (100%) 和敏感性 (83.3%)，且與Bio Chek PRRSV ELISA Kit有正相關的趨勢。未來可用以PRRSV特異性抗體之檢測。

Porcine reproductive and respiratory syndrome (PRRS) infection is one of common diseases in pigs. PRRS infection could cause late-term reproductive failure in sows and severe interstitial pneumonia in weaning pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus in Arteriviridae family. PRRSV genome is 15 kb in size and contains 8 open reading frames (ORFs). In these ORFs, the responsibility the ORF5 gene is to translate 26-30 kDa amount of glycosilation envelope protein (GP5 protein). The goal of this study was developmental a GP5 based ELISA.The ORF5 gene was amplified by RT-PCR. Then the ORF5 gene was constructed into the pET-32a vector, and used the E. coli expression system to express this recombinant protein. The protein was next purified with Ni2+ affinity column and the antigenicity was characterized by western blot and ELISA with anti-His and PRRSV infected swine serum. The effectiveness of the Homemde PRRSV ELISA Kit in this work was compared with commercialized Bio Chek PRRSV ELISA Kit with 56 serum specimens of out-bred farm sows. The PRRSV ELISA revealed high specificity (100%) and sensitivity (86%) and also have positive correlations with commercial Bio Chek PRRSV ELISA Kit. Collectively, the Homemde PRRSV ELISA Kit are potentially used for specific PRRSV antibodies detection in the future.

目 錄
中文摘要................................................ I
Abstract.............................................. II
謝 誌................................................. IV
目 錄................................................... V
圖表目錄................................................. VII
第1章 前 言.............................................. 1
第2章 文獻回顧............................................ 3
2.1豬生殖與呼吸綜合症之歷史................................. 3
2.2豬生殖與呼吸綜合症的命名................................. 3
2.3 豬生殖與呼吸綜合症病毒的特性............................. 3
2.4 豬生殖與呼吸綜合症之臨床症狀............................. 5
2.5 豬生殖與呼吸綜合症病毒之傳播............................. 6
2.6 豬生殖與呼吸綜合症病毒的致病率........................... 7
2.7 豬生殖與呼吸綜合症病毒的致病機制.......................... 7
2.8 豬生殖與呼吸綜合症之體液免疫性反應........................ 7
2.9 豬生殖與呼吸綜合症之細胞免疫性反應........................ 7
2.10 豬生殖與呼吸綜合症的防治............................... 8
2.11 E. coli表現系統..................................... 8
2.12 enzyme-linked immunosorbent assay 之應用............ 9
第3章 材料與方法.......................................... 14
3. 1實驗流程圖........................................... 14
3. 2 重組台灣PRRSV株的ORF5基因............................. 15
3.2.1 MARC-145細胞培養................................... 15
3.2.2 PRRSV之增殖 ........................................15
3.2.3 Total RNA 萃取.................................... 15
3.2.4 PRRSV診斷用引子對................................... 16
3.2.5 反轉錄反應......................................... 16
3.2.6 聚合酶連鎖反應...................................... 17
3.2.7 DNA電泳分析........................................ 17
3.2.8 PCR產物膠體純化回收.................................. 17
3.2.9 接合作用........................................... 18
3.2.10 E. coli DH5α勝任細胞之製備......................... 18
3.2.11 轉形作用（transformation）......................... 18
3.2.12菌落之篩選......................................... 19
3.2.13 DNA定序.......................................... 19
3.2.14 ORF5序列比對...................................... 19
3.3 E. coli表現系統自製GP5檢測ELISA ........................19
3.3.1 設計E. coli表現系統ORF5的引子對...................... 19
3.3.2重組原核表現系統裝載pET32a載體......................... 20
3.3.3 重組原核表現系統裝載pET32a載體確認..................... 20
3.3.4 重組基因轉形到BL21大腸桿菌............................ 20
3.3.5 蛋白質誘導......................................... 21
3.3.6 蛋白質萃取......................................... 21
3.3.7 SDS-PAGE 蛋白質電泳確認pET32a-PRRSV-GP5蛋白.......... 22
3.3.8 西方點墨法確認pET32a-PRRSV-GP5蛋白................... 22
3.3.9 pET32a-PRRSV-GP5蛋白質純化......................... 23
3.3.10 酵素免疫分析法 (ELISA)............................. 24
3.3.11 ELISA抗原及抗體定量試驗 (Box titration)............. 24
第4章 結 果.............................................. 26
4.1 增殖PRRSV........................................... 26
4.1.1 PRRSV感染MARC-145................................. 26
4.1.2 利用PCR確認PRRSV和ORF5............................. 26
4.1.3 ORF5基因序列定序.................................... 26
4.2 利用原核表現系統產生pET32a-PRRSV-GP5次單位蛋白........... 27
4.2.1 PRRS病毒ORF5基因之PCR擴增........................... 27
4.2.2 ORF5基因裝載入yT&;A vector.......................... 27
4.2.3 ORF5基因裝載入pET32a vector........................ 27
4.2.4藉由SDS-PAGE蛋白質電泳分析IPTG誘導之結果 ................27
4.2.5西方點墨法分析pET32a-PRRSV-GP5蛋白之確認............... 28
4.2.6 pET32a-PRRSV-GP5蛋白質純化以西方點墨法分析之結果........ 28
4.2.7 ELISA抗原及抗體定量 (Box titration) 之結果........... 28
4.2.8 BioChek PRRSV ELISA Kit檢測之結果.................. 29
4.2.9 利用ELISA分析感染過PRRSV豬隻血清之靈敏度............... 29
4.2.10 比較BioChek PRRSV ELISA Kit和自製PRRSV ELISA之結果.. 29
第5章 討 論.............................................. 44
參考文獻................................................. 49
附錄.................................................... 60
作者簡介................................................. 71

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