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| 研究生： |
童安廷 Tong, An-Ting |
|---|---|
| 論文名稱： |
豬環狀病毒類病毒顆粒之純化及免疫效力評估 Purification and Immune Evaluation of Porcine Circovirus Type 2 Virus-Like Particles |
| 指導教授： |
柯冠銘
Ke, Guan-Ming |
| 學位類別： |
碩士 Master |
| 系所名稱： |
獸醫學院 - 動物疫苗科技研究所 Graduate Institute of Animal Vaccine Technology |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 81 |
| 中文關鍵詞： | PCV2d 、ORF2 、PCVAD 、桿狀病毒表現系統 、純化 、中和抗體免 疫螢光染色試驗 |
| 外文關鍵詞： | PCV2d, ORF2, PCVAD, baculovirus expression system, purification, immunofluorescence test |
| DOI URL： | http://doi.org/10.6346/NPUST201900115 |
| 相關次數： | 點閱：20 下載：0 |
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豬環狀病毒第II型（Porcine circovirus type II；PCV2）通常感染4至12週齡的豬，引起豬環狀病毒相關性疾病(Porcine circovirus-associated disease；PCVAD)，臨床症狀包括漸進式消瘦、淋巴細胞流失和淋巴結腫大等，及伴有繼發性疾病，在豬場的影響不可輕視。尤其，近年流行之PCV2b和PCV2d兩種基因型具有高致病性。本研究目的為建立ORF2次單位蛋白純化方法並評估其抗原性。本研究以桿狀病毒表現系統生產PCV2d ORF2重組蛋白，經由RNase、Urea與SDS三種不同處理方式後，回收率為10%、32.6%與56.1%，純度為100%、93.2%與89.9%。以80μg的RNase處理之重組蛋白、未純化之重組蛋白，與市售疫苗(Ingelvac CircoFLEX®)進行第一次豬隻實驗。以ELISA分析免疫後血清抗體結果顯示，於免疫後第三週RNase處理組及未純化組的平均抗體力價比市售疫苗組佳(p<0.05)，RNase處理組的平均增重皆比市售疫苗組佳(p<0.05)。但未純化組與市售疫苗的平均中和抗體力價較高，二組之間無顯著差異，而RNase處理組力價較低，與市售疫苗具顯著差異(p<0.05)。第二次豬隻實驗以80μg的Urea處理之重組蛋白、SDS處理之重組蛋白進行。因初次免疫前已證實PCV2自然感染，因此難以判斷ELISA抗體及中和抗體上升之差異，但皆可確認重組蛋白組的抗體提升，並可推測SDS處理組的平均中和抗體力價比Urea處理組高，因此可推測SDS處理之重組蛋白較有效。由兩次動物實驗可得知，未純化組與市售疫苗之中和抗體效果相當，但未純化組在體重等個體表現差於其他處理組的表現，且有副作用之疑慮。考量三者處理方式後，可以用SDS處理重組蛋白之方法，仍保有抗原性可產生良好的中和抗體力價，深具開發疫苗之潛力。
Porcine circovirus type 2 (PCV2) usually infects pigs 4 to 12 weeks of age, causing porcine circovirus-associated disease (PCVAD). Clinical symptoms include marasmus, lymphocyte depletion, and lymphadenopathy. Pigs also often have other concomitant disease infections, and the impact on the farm cannot be underestimated. Among them, PCV2b and PCV2d are popular in recent years and have high pathogenicity. The purpose of this study was to establish an ORF2 subunit protein purification method and evaluate its antigenicity. In this study, we used the baculovirus expression system to produce PCV2d ORF2 protein. After protein treatments by RNase, urea and SDS, the recovery rates were 10%, 32.6% and 56.1%. The purity was 100%, 93.2% and 89.9%. The first animal test was performed with RNase-treated PCV2d ORF2 protein, a commercial vaccine, and an untreated PCV2 ORF2 protein. The average ELISA antibody increase of RNase-treated and untreated groups was better than that of the commercial vaccine group (p < 0.05). Moreover, the average weight gain of the RNase-treated group was better than that of the commercial vaccine group (p<0.05). However, the average neutralizing antibody titers of the untreated group and the commercial vaccine group were higher, and there was no significant difference between the two groups, while the RNase-treated group had a lower titer, which was significantly different from the commercial vaccine group (p<0.05). The second animal test was carried out with a urea-treated recombinant protein and a SDS-treated recombinant protein. Since piglets are confirmed to be naturally infected with PCV2 before the initial vaccination, it is difficult to judge the significant difference between the ELISA antibody and the neutralizing antibody of the recombinant protein. However, it was reconfirmed that the antibody titer of the recombinant protein group was increased, and it was estimated that the average neutralizing antibody titer of the SDS-treated group was higher than that of the urea-treated group, and therefore it was speculated that the recombinant protein treated with SDS was more effective. It can be known from two animal tests that the untreated group is equivalent to the commercial vaccine neutralizing antibody. However, the weight gain of the untreated group is worse than that of the other treatment groups, and there are concerns about side effects. After considering the three treatment methods, the method of treating recombinant protein with SDS is the best option, and the antigenicity can still produce good neutralizing antibody efficacy.
中文摘要 I
Abstract III
謝 誌 V
目 錄 VII
圖表目錄 XI
第1章 前 言 1
第2章 文獻回顧 4
2.1 豬環狀病毒(Porcine circovirus；PCV)研究簡介 4
2.1.1 豬環狀病毒歷史簡介 4
2.1.2 第二型豬環狀病毒之結構與特性 6
2.1.3 第二型豬環狀病毒的感染途徑 7
2.1.4 第二型豬環狀病毒的致病機轉 7
2.1.5 第二型豬環狀病毒對於免疫系統之影響 8
2.1.6 第二型豬環狀病毒之臨床症狀 9
2.1.7 第二型豬環狀病毒的防治 10
2.1.8 第二型豬環狀病毒之疫苗發展及現況 11
2.2 研究目的與動機 12
第3章 材料與方法 14
3.1 研究所使用之病毒株、細菌、細胞與載體 14
3.1.1 Porcine circovirus type 2 (PCV2) 14
3.1.2 Escherichia coli DH-5α 14
3.1.3 pBAC PAK8 14
3.1.4 Spodoptera frugiperda 9 cell (SF-9) 14
3.1.5 High Five cell (Hi-5) 14
3.1.6 Porcine Kidney-15 cell (PK-15) 15
3.2 建構pBac PAK8_PCV2d ORF2重組基因 15
3.2.1 製作pBac PAK8載體 15
3.2.2 萃取pBac PAK8質體 15
3.2.3 pBac PAK8載體限制酶切割作用 15
3.2.4 PCV2d ORF2來源 16
3.2.5 PCV2d ORF2轉型與培養 16
3.2.6 PCV2d ORF2基因片段限制酶切割作用 16
3.2.7 DNA電泳分析 17
3.2.8 PCR產物膠體純化回收 17
3.3 桿狀病毒表現系統表現PCV2d ORF2重組蛋白 17
3.3.1 接合作用 17
3.3.2 轉型作用(transformation) 18
3.3.3聚合酶連鎖反應(Polymerase chain reaction ; PCR)確認 18
3.3.4 質體共轉染 18
3.3.5 昆蟲細胞表現系統 19
3.3.5.1 以昆蟲細胞培養初代病毒 19
3.3.5.2 病毒放大培養於SF-9至p3 19
3.3.5.3 重組桿狀病毒感染Hi-5表現蛋白 19
3.3.6. 以RNase進行不可溶蛋白處理 20
3.3.7 蛋白變性作用 20
3.3.7.1 以Urea進行蛋白變性作用 20
3.3.7.2 以SDS進行蛋白變性作用 20
3.3.8 Urea處理之重組蛋白純化 21
3.3.9 重組蛋白透析 21
3.3.9.1 Urea處理之重組蛋白透析 21
3.3.9.2 SDS處理之重組蛋白透析 22
3.3.10 SDS-PAGE 蛋白質電泳 22
3.3.11 Western blot 23
3.3.12 蛋白質濃度定量 23
3.3.13 電子顯微鏡確認類病毒顆粒 24
3.4 實驗動物設計流程 24
3.4.1 第一次實驗動物組別設定 24
3.4.2 第二次實驗動物組別設定 25
3.5 第一次免疫效力試驗 25
3.5.1 血清抗體反應 25
3.5.2 排毒測定 25
3.5.3 病毒血症測定 26
3.5.4 脾臟組織中PCV2病毒檢測 26
3.5.5 體溫檢測 26
3.5.6 日增重變化測定 26
3.5.7 血清中和抗體免疫螢光染色試驗 26
3.6 第二次免疫效力試驗 27
3.6.1 血清抗體反應 27
3.6.2 病毒血症測定 27
3.6.3 血清中和抗體免疫螢光染色試驗 28
第4章 結果 29
4.1 PCV2 ORF2基因合成 29
4.1.1 PCV2外殼蛋白基因比對 29
4.2 以桿狀病毒表現系統進行PCV2 ORF2重組蛋白生產 29
4.2.1 重組蛋白載體(pBac PAK8)轉染至SF-9結果 29
4.2.2 重組蛋白生產結果 29
4.2.3 重組蛋白沉澱可溶處理 29
4.2.4 純化結果 30
4.2.5 透析結果 30
4.2.6 電子顯微鏡確認類病毒顆粒結果 31
4.3 第一次動物試驗 31
4.3.1 血清抗體反應 31
4.3.2 排毒測定結果 31
4.3.3 病毒血症測定結果 31
4.3.4 脾臟組織中PCV2病毒檢測結果 32
4.3.5 日增重變化測定結果 32
4.3.6 體溫檢測結果 32
4.3.7 血清中和抗體免疫螢光染色試驗結果 32
4.4 第二次動物試驗 33
4.4.1 血清抗體反應 33
4.4.2 病毒血症測定結果 33
4.4.3 血清中和抗體免疫螢光染色試驗結果 33
第5章 討論 66
參考文獻 69
附錄 77
作者簡介 81
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校外公開2024/07/14