利用微生物表達第二型豬環狀病毒衣殼蛋白之研究

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Title:	利用微生物表達第二型豬環狀病毒衣殼蛋白之研究 The study of microbial expression of porcine circovirus type 2 capsid protein
Authors:	林詒卿
Contributors:	生物科技系暨研究所田乃月江建民
Keywords:	第二型豬環狀病毒衣殼蛋白 Porcine circovirus type 2 (PCV2) Capsid protein (CAP)
Date:	2012
Issue Date:	2012-11-29 11:36:32 (UTC+8)
Abstract:	第二型豬環狀病毒 ( Porcine circovirus type 2; PCV2 ) 為引起豬之離乳後多系統消耗性綜合症的主要病原。PCV2 病毒的 ORF2 區域可轉譯出衣殼蛋白 (Capsid protein, CAP)，具有抗原性，可引發宿主產生抗體。以異源表達方式生產 PCV2 CAP 蛋白作為次單位疫苗是目前重要的研究方向。本研究比較了微生物系統表達 PCV2 衣殼蛋白的情形。pET21-6xHis-SUMO-mCAP載體於大腸菌表達系統使用，以方便利用 6xHis來純化、利用 SUMO 提高水溶性及表達量，並利用 SUMO 蛋白酶剪切來移除融合伴SUMO 。大腸桿菌系統研究發現，SUMO-CAP蛋白在 BL21(DE3) 菌株中完全沒有表達；但進一步改用 Rosetta2 (DE3) 菌株表達，獲得了大量表達的 SUMO-CAP 目標蛋白，明顯指出有密碼子的影響。在酵母菌的表達系統，也測試若干融合伴系統，除了 SUMO 蛋白，還包括在酵母菌高效表達的 SFT 蛋白、避免水解的蛋白酶抑制分子 Ecotin 、及方便檢測的 EGFP 。 SFT-CAP蛋白的表達情形，由TLC分析指出 SFT-CAP 胞外的表達量與僅表達 SFT 相比其活性來得較少，沉澱物中有SFT活性也暗示 CAP 蛋白會自行聚集沉澱；由膠體活性分析顯示 SFT-CAP有降解的情形。利用蛋白酶抑制分子Ecotin作為融合伴時，Ecotin-CAP的表達結果在預期位置有明顯條帶，初步結果顯示表達情形較好； EGFP 融合伴可經由螢光以及 Western Blot 方式方便我們分析表達產物，測試結果在 60 kDa 位置有不明顯目標蛋白表達的條帶出現，且在 30 kDa 處也一有明顯多出的條帶產生，我們推測是 EGFP 斷裂的片段。Bacillus胞外及胞內的系統已構築完成正進行表達分析。 Porcine circovirus type 2 is a pathogen related to a disease called porcine post-weaning multisystemic wasting syndrome. The ORF2 of PCV2 gene encodes the capsid protein (CAP), that contains the main antigens to elicit host immunue response. The PCV2 CAP protein produced heterologously is the main target of subunit vaccine development. In this study, several microbial expression systems for CAP protein production were compared. A 6xHis-SUMO-mCAP vector was constructed for E. coli expression, with the ease of purification of CAP protein via Ni2+ chromatography and removal of SUMO fusion part through SUMO protease digestion. The SUMO-CAP fusion protein was successfully expressed in Rosetta 2 (DE3) but not in BL21 (DE3), indicating the codon usage bias. Several vectors using fusion protein system for extracellular expression of CAP protein in Pichia pastoris were constructed and tested, including the highly soluble SFT, protease inhibitor Ecotin, and EGFP, to ficilitate expression, tracing, or preventing degradation. The results of direct CAP protein expression, as well as SUMO-CAP expression, showed insignificant protein content in the culture broth. However, SFT-CAP was expressed a little bit better. The result of SDS-PAGE combined with in gel assay indicated that SFT-CAP was degraded through the expression. The presence of SFT activity in the precipitate indicated the aggregation of SFT-CAP protein. The extracellular expression level of Ecotin-CAP was the best within the fusion protein tested. Vectors for intracellular and extracellular expression of CAP protein in the bacillus system were constructed. The expression has been put into progress.
Relation:	校內校外均不公開，學年度：100，58頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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