- 學位論文
豬環狀病毒第二型疫苗研發技術之研究
Studies on the Vaccine Technology Development of Porcine Circovirus Type 2
摘要
豬環狀病毒第二型(PCV2)引起的相關疾病(PCVAD)，對全世界的養豬產業造成很大的經濟損失。防治PCVAD的方法包括生物安全防護、合宜的牧場管理、以及疫苗注射。目前市售三種商品化的豬環狀病毒第二型疫苗，包括不活化全病毒疫苗、不活化PCV1載體帶PCV2疫苗、與PCV2次單位疫苗。PCV2病毒無法在豬腎臟細胞PK-15中造成細胞病變效應(CPE)，檢測病毒需依賴螢光染色，因此本論文建構以帶有加強型綠螢光蛋白(enhanced green fluorescent protein; EGFP)的PCV2，可做為檢測PCV2病毒力價或測定中和抗體時的良好工具。本論文之另一個重要目的是藉由各種疫苗開發技術提升，來增進PCV2疫苗的效果。其中包含次單位疫苗、DNA疫苗、以及全病毒疫苗。本論文第一部份，將全長豬環狀病毒第二型基因體與加強型綠螢光蛋白基因融合，並構築在pBluescript (pSK)載體上，送入細胞，試圖產製出攜帶綠螢光蛋白的PCV2。結果顯示，於5個PCV2 ORF中，只有在ORF2下游接上EGFP基因後，能順利在PK-15細胞核中產生綠色螢光，並且進行病毒複製。反之，其他的構築，包括PCV2 (ORF1)-EGFP/pSK、PCV2 (ORF3)-EGFP/pSK、PCV2 (ORF4)-EGFP/pSK及PCV2(ORF5)-EGFP/pSK，轉染後之細胞均無出現特異螢光。細胞轉染後所轉譯出的ORF2-EGFP蛋白質，以PCV2抗體進行免疫螢光染色，可同時偵測到綠色及rhodamine標示的紅色螢光。此外，攜帶綠螢光蛋白的PCV2 DNA，自細胞釋放出後，可用即時定量聚合酶鏈鎖反應(real-time PCR)定量分析，並可自連續繼代二次後的上清液中檢測出來。本研究第二部份，利用去毒化綠膿桿菌外毒素A(Detoxified PE)，來增強PCV2 ORF2重組蛋白之免疫抗原性。首先將五個不同片段的PCV2 ORF2之抗原區基因，進行選殖及以大腸桿菌表現大量的蛋白，並以大鼠評估各重組蛋白引發的免疫原性。結果顯示，PCV2 ORF2 F2片段(氨基酸序列第78-156區塊)具有最佳的免疫原性。將去毒綠膿桿菌外毒素A基因及KDEL訊號序列基因與F2基因融和(稱PE-F2-KDEL重組蛋白)，來評估應用PE的結合及轉位區塊作為攜帶PCV2 ORF2 F2免疫抗原的效果。本實驗分別將F2及PE-F2-KDEL重組蛋白進行小鼠的腹腔免疫注射。結果顯示，PE-F2-KDEL有顯著性地，比F2更能誘發較佳的抗PCV2血清IgG抗體力價。此外，與PCV2不活化病毒抗原比較，PE-F2-KDEL重組蛋白亦有顯著性地提高抗PCV2血清IgG的力價。本實驗證實，去毒綠膿桿菌外毒素A可誘發PCV2 ORF2重組蛋白較強的免疫反應。本論文的第三個目標，乃開發一個多重功能之融合蛋白來搭配PCV2 DNA疫苗，以協助DNA分子送入體內細胞。由於DNA疫苗進入體內和細胞內的傳送效率低，所以DNA疫苗在臨床及實際運用上非常侷限。此研究中的多重功能之融合蛋白包VP22-TmHU-PCV2.NLS含源自單純皰疹病毒的VP22胜肽，Thermotoga maritima的DNA結合蛋白HU(TmHU)以及PCV2 ORF2之核定位訊號片段(NLS)。首先，以洋菜膠電泳延阻試驗(electrophoretic mobility shift assay; EMSA)進行測試結果：VP22-TmHU(VT)及VP22-TmHU-PCV2.NLS (VTN)都能有效率的與質體DNA (pEGFP-N1)結合。隨後，經由轉染pEGFP-N1的細胞，分別以螢光顯微鏡和流式細胞儀觀察及分析pEGFP-N1細胞內之傳遞效率，結果發現，VTN可協助pEGFP-N1順利傳遞到細胞內，但VT則不能直接攜帶DNA 轉染於細胞內。若分別將VT與VTN加入Lipofectamine™，經轉染測試顯示；二者都能增加Lipofectamine™的傳遞效率，由原本單獨使用Lipofectamine™時的25%增加到65%。在動物體內試驗時，在所有組別裡，包括PBS、pcDNA3-ORF2、pcDNA3-ORF2+Lipofectamine、pcDNA3-ORF2+VT、pcDNA3-ORF2+VT+Lipofectamine、pcDNA3-ORF2+VTN、和pcDNA3-ORF2+VTN+Lipofectamine，結果以pcDNA3-ORF2+VTN組，能誘導出最高抗PCV2 ORF2外鞘蛋白的抗體。與體外試驗不同的是，VTN混合Lipofectamine™之後，並沒有增強抗PCV2 ORF2的抗體力價。依據以上結論，PCV2 ORF2的NLS可增進DNA質體進入哺乳類細胞的傳送。本論文最後一部份乃建立了一個高感受性及潔淨的細胞株來產製PCV2。從一株PCV1污染的PK-15，以高免兔抗PCV1抗體，經三次淨化處理，並經過極限稀釋單株化後，挑選出四個單一細胞株，經單層培養後，進行PCV2增殖測試。結果顯示，其中一株PKKC細胞最為敏感並適合用來增殖高力價PCV2。PKKC細胞感染PCV2六天後，病毒力價可達106.8 TCID50/ml。此外，PKKC細胞亦無其它外源性病毒污染。本細胞感染PCV2後，不但能增殖出高力價的PCV2病毒，也具有產生細胞病變效應(CPE)的特性。本細胞具有PCV2之病毒增殖、疫苗產製、及診斷應用的潛能。
並列摘要
Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus type 2 associated diseases (PCVAD). PCVAD causes huge economical losses in swine productions worldwide. Preventive methods of PCVAD include biosecurity, proper pig farm management, and vaccination. Up-to-date, there are four companies marketing three kinds of commercialized PCV2 vaccines which are PCV2 subunit vaccines, killed PCV1-2 vectored vaccine, and whole viral inactivated vaccine. Since PCV2 does not cause cytopathic effect (CPE) in PK-15 cells, thus detection of PCV2 has relied on immunofluorescence assays (IFA). In this thesis, creating an enhanced green fluorescent protein (EGFP) fused PCV2 could serve as a convenient diagnostic tool for measuring PCV2 titer or neutralizing antibodies. Another major purpose of this thesis was to enhance PCV2 vaccines through various developments in vaccine technology including the improvement of subunit vaccine, DNA vaccine, and full virus inactivated vaccine. In the fist study, construction and transfection of EGFP-fused PCV2 genome in pBluescript (pSK) vector and the recovery of the virus was described. When fusing EGFP downstream of five open reading frames (ORF) of PCV2, green fluorescent signals were observed in the nucleus of PK-15 cells only after PCV2 (ORF2)-EGFP/pSK transfection while PCV2 (ORF1)-EGFP/pSK, PCV2 (ORF3)-EGFP/pSK, PCV2 (ORF4)-EGFP/pSK, and PCV2(ORF5)-EGFP/pSK showed no fluorescent signals post-transfection. The presence of ORF2-EGFP fusion protein was demonstrated by dual signals of green fluorescence and anti-PCV2 antibodies conjugated with rhodamine in immunofluorescence assay (IFA). Furthermore, the released EGFP-fused PCV2 genome was quantified by real-time PCR for two more passages. In the second study, five different fragments of PCV2 ORF2 antigenic regions were cloned, over-expressed in E. coli, and immunized in rats to evaluate the immunogenicity of the PCV2 ORF2 recombinant proteins. Results showed that the ORF2 F2 fragment (residues 78-156) was the most immunogenic in terms of the antibody induction. Detoxified Pseudomonas exotoxin A (PE) and KDEL signal peptide were fused with F2 fragment at the N and C terminus, respectively. This study evaluated the application of using the binding and translocation domains of PE as a vehicle for PCV2 F2 fragment, resulting in the construction of a PE-F2-KDEL recombinant protein. F2 and PE-F2-KDEL recombinant proteins were intraperitoneally inoculated in mice, respectively. Results showed that PE-F2-KDEL induced significantly higher anti-PCV2 serum IgG antibodies than F2. Additionally, PE-F2-KDEL recombinant protein also stimulated significantly higher PCV2-specific IgG response compared to inactivated PCV2 whole virus antigen. These results showed that detoxified Pseudomonas exotoxin A could enhance the immune response of a PCV2 ORF2 recombinant protein. The third goal of the thesis was to construct and express a multiple function fusion protein to enhance intracellular delivery for PCV2 DNA vaccine. DNA vaccines are limited in clinical and practical uses due to its ineffective delivery. In this research study, the multiple function protein VP22-TmHU-PCV2.NLS was composed of cell-penetrating peptide originated from VP22 peptide of herpes simplex virus, DNA binding HU protein from Thermotoga maritima (TmHU), and a PCV2 nuclear localization signal (NLS). Firstly, as shown by the electrophoretic mobility shift assay (EMSA), VP22-TmHU (VT) and VP22-TmHU-PCV2.NLS (VTN) were able to bind to plasmid DNA (pEGFP-N1) effectively. Secondly, intracellular transport of pEGFP-N1 was observed by fluorescence microscopy and quantified by flow cytometry after transfection. VTN was successful in delivering pEGFP-N1 intracellularly but VT was not. Thirdly, when combined with Lipofectamine™, both VT and VTN enhanced the transfection rate from 25% with Lipofectamine™ alone up to 65%. Lastly, mice were injected intramuscularly with PBS, pcDNA3-ORF2, pcDNA3-ORF2 plus Lipofectamine™, pcDNA3-ORF2 plus VT, pcDNA3-ORF2 plus VT plus Lipofectamine™, pcDNA3-ORF2 plus VTN, and pcDNA3-ORF2 plus VTN plus Lipofectamine™. The highest level of antibodies raised against PCV2 ORF2 Cap protein was detected with pcDNA3-ORF2 plus VTN. Contrary to the in vitro results, VTN combined with Lipofectamine™ did not further enhance antibody level against PCV2 ORF2. In summary, the NLS of PCV2 ORF2 can enhance intracellular delivery of plasmid DNA. The last study of this thesis established a highly permissive and decontaminated cell line for growing porcine circovirus type 2 (PCV2). A porcine kidney-15 cell line (PK-15) contaminated with porcine circovirus type 1 (PCV1) was decontaminated by neutralizing with rabbit anti-PCV1 hyperimmune serum. Subsequently, by limiting dilution and cell subcloning, four PCV1-free monoclonal cells were grown to monolayers. Each cell subclones and PK-15 cell were infected with PCV2. The PKKC cell clone yielded up to 106.8 TCID50/ml at six days post-infection. In addition, PKKC was free of extraneous viral contamination and exhibited a cytopathic effect (CPE) to PCV2 at six days post-infection. The advantages of the PKKC cell are that it can grow a high PCV2 titer and exhibit CPE; therefore, it can be used for PCV2 cultivation, vaccine production, and diagnostic purposes.