| 摘要: | 先前研究證實傳染性華氏囊病病毒(infectious bursal disease virus, IBDV)VP2-452H蛋白自我組裝形成的次病毒顆粒，能以Ni-NTA親和管柱單一步驟純化。但經由VP2-452H次病毒顆粒之X-ray晶體繞射分析與酵素連結免疫分析法，證實VP2-452H蛋白所融合之His-tag並未暴露於次病毒顆粒表面，故推論VP2-452H次病毒顆粒可被固定化金屬離子親和層析純化之原因，是本身顆粒表面即具能與固定化金屬離子形成作用力。為此以Ni-NTA管柱純化VP2蛋白構成病毒外鞘之IBDV病毒與未融合His-tag的重組VP2-441次病毒顆粒。純化結果為IBDV病毒回收率達60.5%，而次病毒顆粒的純度與回收率達92%。再者藉由穿透式電子顯微鏡之觀察，證實分別純化IBDV與VP2-441次病毒顆粒後的樣品中，有粒徑為65 nm的IBDV病毒顆粒與25 nm的次病毒顆粒粒子。証實IBDV病毒表面能與固定化金屬離子形成親和力之特性。然而本研究以單點突變技術更進一步解釋病毒表面主要由哪種胺基酸構成病毒顆粒被Ni-NTA管柱純化，VP2-441胺基酸序列中的His249與His253經置換為Ala後，所表現的重組次病毒顆粒是無法以Ni-NTA管柱純化。由上述實驗結果證實IBDV VP2蛋白胺基酸序列中之His249與His253能使蛋白在組裝後的病毒顆粒具有與固定化鎳離子產生親和力的特性，故能幫助IBDV與次病毒顆粒藉由Ni-NTA管柱純化。本研究以恆溫吸附實驗解釋病毒顆粒表面是以多個His253，與固定化金屬離子形成親和力，使次病毒顆粒能被有效純化。首先以恆溫吸附曲線圖比較出VP2-441次病毒顆粒對Ni-NTA樹脂的吸附力高於表面帶有2-3個histidine之BSA蛋白，說明次病毒顆粒表面需提供數個His253增進顆粒對樹脂之吸附力。再者，次病毒顆粒與Ni-NTA樹脂間產生的異相吸附行為與BSA蛋白不同，由Scatchard plot分析恆溫吸附圖形，顯示VP2-441次病毒顆粒之曲線走勢呈現concave up的屬性，而BSA蛋白為concave down。此外發現單位體積Ni-NTA樹脂對次病毒顆粒的飽和吸附量低於BSA蛋白近400倍。為此本研究以共軛焦顯微鏡分析觀察樹脂吸附次病毒顆粒後之螢光分布，顯示只有在樹脂表面有螢光散布，意指次病毒顆粒不易進入孔洞過小的樹脂內進行吸附，顯示目前使用之Ni-NTA樹脂並不能有效地吸附次病毒顆粒。最後次病毒顆粒與BSA蛋白之恆溫吸附曲線以Langmuir-Freundlich模式進行適配，顯示VP2-441次病毒顆粒對Ni-NTA樹脂的吸附力高於BSA蛋白300倍，証實VP2-441次病毒顆粒對Ni-NTA樹脂有較強的吸附力。VP2-441次病毒顆粒的concave up的曲線屬性，可經由Temkin模式適配，顯示KT值為5×1010 M-1，意指兩者之間的吸附力近似抗體與抗原間之作用力。 The protein VP2, matured from the polyprotein, which was encoded by the genome of infectious bursal disease virus (IBDV), is the primary host-protective immunogen of IBDV. According to VP2-452H subviral particle (SVP) analysis, the determination crystal structure and enzyme-linked immunosorbent assay (ELSIA), showed thar His-tag was not exposed on the surface of VP2-452H SVPs. Thus illustrate that the His-tag apparently did not attribute to the effective purification of this protein by IMAC. An affinity must have existed between the protein VP2 and the immobilized metal ions to achieve SVP binding with Ni-NTA resin. Accordingly, the IBDV generated from DF-1 cell culture and non-tagged VP2-441 SVP generated from a baculovirus-insect cell expression system were purified by IMAC. The purification of IBDV viron through IMAC obtained a 60.5% recovery, and the IMAC-purified IBDV has a similar morphology to the wild-type IBDV with a diameter of 65 nm through electron microscope observation. For SVP formed by VP2-441 purified by IMAC a recovery 92% and a purity of also 92% of mature VP2 were obtained. SVP formed by VP2-441 exhibited a diameter approximately 25 nm. These results obtained from the above experiments can demonstrate 1) the protein VP2 does have interaction with immobilized nickel ion; and 2) the protein VP2 can assist both IBDV viron and SVPs to have the affinity with Ni-NTA resin. The recombinant protein VP2-441, i.e., a structural protein VP2 of infectious bursal disease (IBD) virus, can self-assemble into T=1 subviral particles (SVPs) in baculovirus expression system. These SVPs are not to have multiple his-tags on the surface which result in an efficient purification by immobilized metal-ion affinity chromatography (IMAC). This study aimed at getting more insight into the interaction between VP2-441 SVPs and immobilized metal (Ni2+) ions at molecular level. First of all, large quantity of highly purified VP2-441 SVPs obtained by a one-step purification process allowed the performance of equilibrium adsorption measurements and subsequent determinations of binding constants by fitting two isotherm models, i.e., Temkin and Langmuir-Freundlich. Two general conclusion are obtained, first, the maximum bound VP2-441 SVPs per volume resin is limited because the pore size of IMA gel (ca. 24 nm in diameter) is similar to that of SVPs (20 – 25 nm) and the diffusion of the latter into the pores is hindered. The other is that SVPs have an extremely high affinity to the immobilized Ni+2 ions because the dissociate constants obtained from different models are in the scale of 10-9 M, which suggested the interaction mimicking that between an antigen and its antibody. The high binding strength is derived from a multiple-site binding between VP2-441 SVPs and Ni2+ ions as demonstrated by a concave-up Scatchard plot. Finally, we found that the adsorption of SVPs can be well described by Temkin model. A detail understanding of SVP-immobilized metal ion interactions can provide useful strategies for conducting preparative-scale separations of SVPs or even a real virus using IMAC. |
