臺灣博碩士論文加值系統

細胞轉導胜肽能協助親水性的大分子通過細胞膜，並已應用在各方面，如：細胞轉染、癌症治療、DNA疫苗開發等。豬環狀病毒第二型所引發的離乳後多系統消耗性綜合症被視為是影響豬隻產業的重大疾病之一，目前商品化疫苗多為次單位疫苗及死毒疫苗。上述疫苗主要是以體液性免疫來減少豬隻感染後造成的傷害，但難以清除病原。DNA疫苗可同時誘發出細胞性免疫及體液性免疫。本研究是以細胞轉導胜肽為基礎來提升PCV2 DNA疫苗之免疫效力。結果證明，本論文設計之細胞轉導胜肽可將綠螢光基因pEGFP-N1轉染至中國倉鼠卵巢細胞(CHO cell)中表現出綠螢光蛋白，證實其具有核酸轉導之能力。小鼠模式的免疫實驗中，發現僅DNA疫苗佐以細胞轉導胜肽免疫小鼠後，無法誘導抗體生成，但是若補強注射微量的不活化病毒抗原，則在抗體生成、中和抗體以及淋巴細胞增生上，都比僅施打DNA疫苗的組別及僅施打不活化病毒抗原快且高。本研究證實本研究所設計的兩種核酸轉導胜肽，確實能增強小鼠對於PCV2 DNA疫苗所帶來的免疫效力。

Many kinds of hydrophilic large molecules could be delivered into cells by cell-penetrating peptides (CPPs). CPPs had been used in many kinds applications, including cell transfection, cancer-targeting therapy and DNA vaccine, and so on. Porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), is one of the most important diseases affecting swine industry. The commercial PCV2 vaccines are either inactivated or subunit vaccine. These vaccines can induce humoral immunity to against the injury of virus, but the circulating PCV2 cannot be cleaned. Since DNA vaccine can induce both humoral and cell-mediated immunity, in this study, the cell-penetrating peptide was applied to improve the efficacy of PCV2 DNA vaccine. The cell-penetrating peptides designed in this study are able to deliver pEGFP-N1 into China hamster ovary (CHO) cell and the GFP protein will be expressed. The CPP-DNA vaccine cannot induce anti-PCV2 antibody; however, it will result in higher level of anti-PCV2 antibody, neatralizing antibody and PCV2-induced lymphocyte proliferation when booster with low dose inactivated PCV2 antigen in mice model. These results demonstrate that CPP can enhance the efficacy of PCV2 DNA vaccine via humoral and cell-mediated immunity.

第一章、 前言 - 5 -
第二章、 文獻探討 - 6 -
2.1、豬環狀病毒歷史 - 6 -
2.2、PCV2與相關疾病介紹 - 6 -
2.2.1、生殖障礙 - 7 -
2.2.2、離乳後多系統消耗性綜合症 - 7 -
2.2.3、豬皮膚炎腎病症候群 - 7 -
2.2.4、豬呼吸道疾病綜合症 - 7 -
2.3、PCV2疫苗開發 - 8 -
2.3.1、Circovac(Merial) - 8 -
2.3.2、Ingelvac CircoFLEX® (Boehringer Ingelheim)、 Circumvent (Intervet/Merck)、Porcillis PCV (Schering-Plough/Merck) - 8 -
2.3.3、Fostera™ PCV (Fort Dodge Pfizer) - 8 -
2.4、DNA疫苗介紹 - 9 -
2.5、DNA疫苗誘導細胞免疫相關機制 - 9 -
2.6、動物DNA疫苗之開發 - 10 -
2.7、DNA疫苗免疫方式之策略 - 10 -
2.7.1、物理性傳遞-基因槍 - 10 -
2.7.2、物理性傳遞-超聲波 - 10 -
2.7.3、物理性傳遞-電穿孔 - 10 -
2.7.4、非物理性傳遞-病毒載體 - 11 -
2.7.5、非物理性傳遞-微脂粒 - 11 -
2.7.6、非物理性傳遞-陽離子聚合物(多醣) - 11 -
2.7.7、非物理性傳遞-細胞轉導胜肽 - 11 -
2.8、細胞轉導胜肽應用於DNA疫苗 - 12 -
2.8.1、細胞轉導胜肽發現 - 12 -
2.8.2、細胞轉導胜肽機制 - 12 -
2.8.3、細胞轉導胜肽傳遞DNA - 12 -
2.9、構築PCV2 DNA疫苗 - 13 -
2.10、構築核酸轉導胜肽 - 14 -
2.11、優化DNA分子與核酸轉導胜肽結合之作用環境 - 15 -
第三章、 實驗策略 - 16 -
第四章、 材料與方法 - 17 -
4.1、構築PCV2 DNA疫苗 - 17 -
4.1.1以PCR法增幅Gaussia luciferase + HBc148 + rPCV2片段 - 17 -
4.1.2以洋菜膠電泳分析PCR增幅結果 - 17 -
4.1.3純化PCR產物 - 17 -
4.2、構築Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1 重組載體 - 18 -
4.3、接合反應 - 18 -
4.4、勝任細胞的製備 - 18 -
4.5、轉形作用 - 19 -
4.6、重組質體的挑選與確認 - 19 -
4.7、大量製備質體DNA - 19 -
4.8、人工合成細胞轉導胜肽 - 20 -
4.9、SDS-PAGE確認合成胜肽之大小 - 20 -
4.10、蛋白質定量 - 20 -
4.11、洋菜膠電泳延阻試驗 - 21 -
4.12、細胞轉導胜肽細胞轉導效率分析 - 21 -
4.12.1對照組 - 21 -
4.12.2轉染pEGFP-N1(1× DPBS) - 21 -
4.12.3轉染pEGFP-N1 (10 mM CaCl2) - 21 -
4.12.4轉染Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1組(1× DPBS) - 22 -
4.12.5轉染Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1組(10 mM CaCl2) - 22 -
4.13、以免疫螢光染色法確認DNA疫苗可於轉染細胞內表現抗原 - 22 -
4.14、以流式細胞儀分析DNA質體之轉染效率 - 23 -
4.15、小鼠免疫試驗 - 23 -
4.15.1 DNA疫苗配製 - 23 -
4.15.2 小鼠免疫日程 - 23 -
4.15.3 以酵素連結免疫吸附分析法檢測小鼠抗PCV2之抗體 - 24 -
4.15.4中和抗體力價(VNT90)判讀 - 24 -
4.15.5小鼠脾臟細胞增生試驗 - 25 -
第五章、 結果 - 26 -
5.1、構築PCV2 DNA疫苗 - 26 -
5.2、 SDS-PAGE分析合成胜肽 - 26 -
5.3、細胞轉導胜肽與DNA疫苗結合能力測試 - 26 -
5.4、細胞轉導胜肽轉染DNA疫苗效率測試 - 27 -
5.5、以ELISA檢測小鼠免疫DNA疫苗後之血清抗體力價 - 27 -
5.6、分析小鼠血清中抗PCV2 的中和抗體力價 - 27 -
5.7、以PCV2不活化病毒刺激特異性脾臟細胞增生 - 27 -
第六章、 討論 - 28 -
第七章、 附錄 - 32 -
第八章、 參考文獻 - 52 -
表目錄

表一、選殖DNA疫苗之特異性引子序列 - 32 -
表二、合成胜肽之胺基酸序列 - 32 -
表三、細胞轉導胜肽細胞轉導效率分析之組別及配方 - 32 -

圖目錄

圖一、理想的DNA疫苗免疫機制 - 33 -
圖二、至2013年為止，於市面上發行的四種DNA疫苗 - 34 -
圖三、細胞穿透胜肽幫助核酸進入細胞膜及核膜示意圖 - 34 -
圖四、構築DNA疫苗Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1示意圖 - 35 -
圖五、以PCR法增幅PCV2 ORF2 片段之電泳分析圖 - 35 -
圖六、酵素切割確認 (Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1) - 36 -
圖七、DNA疫苗(Gaussia luciferase + HBc148 + rPCV2 dNLS/pVAX1)定序結果 - 39 -
圖八、分析胺基酸大小 - 40 -
圖九、以EMSA 法測試合成胜肽Antp-Mu-NLS與DNA之結合能力 - 41 -
圖十、以EMSA 法測試合成胜肽Antp-Mu與DNA之結合能力 - 42 -
圖十一、以EMSA 法測試合成胜肽TAT-Mu-NLS與DNA之結合能力 - 43 -
圖十二、以EMSA 法測試合成胜肽TAT-Mu與DNA之結合能力 - 44 -
圖十三、合成胜肽可促進DNA分子進入目標細胞之分析圖(A) - 45 -
圖十四、合成胜肽促進DNA分子進入真核細胞並表現抗原之分析圖(A) - 46 -
圖十五、合成胜肽佐以氯化鈣可促進DNA分子進入目標細胞之分析圖(B) - 47 -
圖十六、合成胜肽佐以氯化鈣促進DNA分子進入真核細胞之分析圖(B) - 48 -
圖十七、BALB/c小鼠DNA疫苗及PCV2不活化疫苗補強免疫日程 - 48 -
圖十八、以ELISA法檢測免疫後小鼠體內抗PCV2之特異性抗體力價 - 48 -
圖十九、免疫後小鼠體內抗PCV2之中和抗體力價分析圖 - 51 -
圖二十、免疫後小鼠脾臟以增生試驗分析圖 - 51 -

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