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研究生:鄭宇伸
論文名稱:二階段式色層分析程序應用於傳染性華氏囊病病毒似病毒粒子純化分離之程序設計及研究
論文名稱(外文):Separation of Pure and Immunogenuc Virus-Like Particles Using Gel-Filtration Chromatography Following Immobilized Metal Affinity Chromatography
指導教授:王敏盈
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:77
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由昆蟲細胞-桿狀病毒表現系統(Insect cell culture / baculovirus expression
system)所表現C端接有6個組氨酸(Histidine)之傳染性華氏囊病病毒(infectious
bursal disease virus;簡稱IBDV)的主要結構蛋白單體(rVP2H),所形成之似病毒粒子
(Virus-like particlse;簡稱VLPs)經證實已可以有效防止IBDV感染雞隻,為了進一步
了解其抗原結構及開發成疫苗的可行性,必需取得大量高純度之rVP2H particles。但目
前仍無一有效分離程序可大量將生產過程中所產生的rVP2H及VLPs分離。本研究主要發展
出固定化金屬離子親和性色層分析法(Immobilized metal-ion affinity
chromatography, IMAC)合併凝膠過濾色層分析法(Gel filtration chromatography)的二
段式純化程序,來大量分離純化經由細胞培養生產的rVP2H及VLPs,藉由SDS-PAGE及酵素
連結免疫吸附測定(Enzyme-linked immunoabsorbent asssay)證明利用此二段式純化程序
可以有效分離rVP2H及VLPs兩種型態之重組蛋白,分離所得之VLPs經由穿透式電子顯微鏡
及氯化銫梯度離心顯示粒徑約為20nm,浮力密度1.27g/mL,同時純度更達90%以上,未來
可進一步將此一程序放大應用於類似蛋白的大量分離約化。
A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of infectious bursal disease virus (IBDV) with additional six histidine residues at its C-terminus, for the study of its antigenicity and conformational structure and the development of an efficient subunit vaccine against IBDV infection. The expressed VLPs mimic th authentic structure of the critical epitopes in the native viruses and are capable of inducing a stronger immunological response than does the free form of rVP2H. Those particles in the inected High-FiveTM cell lysates were partially purifed employing immobilized metal-ion (Ni+2) affinity chromatography (IMAC). This step recovered about 85 percent of immunogeric rVP2H proteins but did not separate the rVP2H particles from the free rVP2H proteins or the degraded products of rVP2H. A further separation of the particulate form of rVP2H from the free form of rVP2H was achieved using either gel filtration chromatography or CsCl density gradient ultracentrifugation. The latter method is suitable for the purification of small amount of rVP2H particles and the particle's buoyant density determine using this method was very close to 1.27 g/cm3. However, when large quantities of the rVP2H particles are needed, it is more efficient to purify the particles by using gel filtration chromatography than the conventional ultracentrifugation method. The separation of the particulate form from the free form of rVP2H proteins enables the evaluation of their own immunogenicity to induce the virus-meutralizing antibodies leading to the protection of chickens from IBDV infection. In addition, the abundant quantities of pure rVP2H particles coupled with the uniform dimensions of the particles will facilitate an examination of higher order structure of the immunogenic particles and, therefore, the design of better vaccines against the virus will evolve.

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