第二型豬環狀病毒重組ORF2與感染性質體的分子選殖

Molecular Cloning of Recombinant ORF2 and Infectious Clone of Porcine Circovirus Type II

邱芮瑜(Jui-Yu Chiu)

指導教授 ： 郭應誠

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10407

摘要

豬離乳後多消耗性症候群 (PMWS)的發生雖然是多因子性，但第二型豬環狀病毒 (PCV2)為主要元兇。PCV2的感染是世界性的，在台灣PCV2抗體盛行率高達八成以上。PCV2的ORF2為外鞘蛋白 (capsid protein)，含有中和抗原決定位而能引起中和抗體。全長ORF2的次單位疫苗與DNA疫苗在實驗感染的豬隻上測試顯示，次單位疫苗比DNA疫苗有較好的保護效力，迄今卻未見有疫苗上市，推測可能是ORF2的重組蛋白質量產與純化上的困難所致。利用原核系統成功表現全長ORF2並不容易，本論文將全長ORF2 (ORF2F)與含較高抗原性區域片段分前、中、後縮短的ORF2片段，稱為ORF2N、ORF2M與ORF2C於大腸桿菌中表現，只有ORF2M、ORF2C能成功表現並純化出重組蛋白，且純化量高達約20 mg/L，所製備的小鼠抗血清及單株抗體，不但能辨識病毒抗原，也具中和抗體力價，其中，ORF2M血清可達到32倍。據此，本論文所製備的ORF2M有潛力作為PCV2次單位疫苗之素材。本論文也嘗試利用真核原蟲表現系統表現ORF2，卻無法大量表現。
PCV2常伴隨其他病原存在動物體中，增加單獨分離此病毒的困難度，再者分離的PCV2病毒在細胞培養中並不易增殖純化。本論文構築具感染性PCV2質體，利用細胞培養建立PCV2量產模式，作為穩定單純的病毒來源。同時觀察病毒在PK-15細胞中增殖時，幾種主要病毒蛋白的表現情況，以及他們對病毒複製能力的影響。結果顯示，病毒感染PK-15細胞後12小時，以西方墨點法偵測細胞質或細胞核都能發現ORF2蛋白的存在，但此時ORF1蛋白則尚未表現，直到感染後24小時才稍見出現，ORF2似乎較ORF1早被表現。另外，ORF1、ORF2及ORF3蛋白的過度表現不顯著影響每一代病毒的增殖，然而ORF2的過多表現可能促成CPE的提早出現；反之，ORF1過多表現時卻延後CPE的出現，因而讓細胞能多繼代2代，生產更多的病毒液。本結果可作為量產PCV2病毒細胞模式的參考條件。

關鍵字

第二型豬環狀病毒 ； 外鞘蛋白 ； 複製相關蛋白 ； 次單位疫苗 ； 感染性質體 ； 重組蛋白

並列摘要

Post-weaning multisystemic wasting syndrome (PMWS) becomes a major problem in many pig-producing countries throughout the world. Porcine circovirus type 2 (PCV2) has been identified as an essential component of PMWS. Approximately 83.5% of the pigs in Taiwan were seropositive to PCV2. The open reading frame 2 (ORF2) encoding the major capsid protein with epitopes to induce neutralizing antibodies is the potential target gene for vaccine development in against PCV2 infection. However the difficulty to effectively express ORF2 seems to limit the development of PCV2 vaccine. In this study, we expressed full-length, N-terminal, middle and C-terminal ORF2 covering high antigenic epitopes by E. coli. Only ORF2M and ORF2C could be overexpressed and purified. The yield of purified ORF2M or ORF2C was about 20 mg/L. Mouse antisera and monoclonal antibodies produced by ORF2M or ORF2C recognized PCV2 under Western blotting and immunofluorescence assay. The neutralization titer of ORF2M-induced serum was about 32X, suggesting that ORF2M is a potential candidate of PCV2 subunit vaccine. We also tried but failed to express ORF2 in protozoa expression system. It is hard to isolate PCV2 from field sample and to propagate it in cell culture. We constructed the infectious clone of PCV2 to generate a biologically pure and homogeneous infectious virus stock to study viral behaviors. In PCV2-infected PK-15 cells ORF2’ was detected at 12 hours post inoculation (h.p.i.) by Western blotting, but ORF1 was detected at 24 h.p.i.. The expression of ORF2 seemed to be earlier than that of ORF1. On the other hand, overexpression of ORF1, ORF2 or ORF3 had no effect on viral propagation. However, overexpression of ORF2 advanced, but ORF1 delayed the appearanceof cytopathic effect (CPE). The ORF1-delayed CPE increased the life span of PCV2-infected cells and then viral propagation. The results provide useful information for efficiently producing PCV2 virus stock in PK-15 cell model.

並列關鍵字

porcine circovirus type II ； capsid protein ； Rep protein ； subunit vaccine ； infectious clone ； recombinant protein

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