傳染性雞華氏囊病毒的VP2蛋白於大腸桿菌中組裝成次微米級似病毒顆粒之研究

Abstract

本研究是將傳染性雞華氏囊病毒的結構蛋白VP2基因構築於表現載體中，利用大腸桿菌表現VP2及C端截切的VP2蛋白，經由不同條件測試，探討其是否能自我組裝形成似病毒顆粒（virus-like particle, VLP）。大腸桿菌表現未帶有任何融合蛋白的全長erVP2可溶蛋白，經氯化銫密度梯度離心，於電子顯微鏡下觀察可見粒徑大小約為20 nm次微米級似病毒顆粒。然而在C端融合了六個組胺酸（6 histidine residues）的erVP2H的可溶蛋白在大腸桿菌BL21(DE3) CodonPlus-RP表現量不高，雖然可以藉由固定化金屬離子色層分析法純化，在電子顯微鏡的觀察下其所形成的為15 nm似病毒粒子。VP2蛋白在大腸桿菌中形成似病毒顆粒的研究未曾被報導，因此本實驗繼續利用此系統表現另外兩個C端截切的VP2蛋白─er1167H及er1197H，並於C端也融合了六個組胺酸，但這兩個不同長度C端截切及融合六個組胺酸的VP2蛋白則未見有似病毒顆粒的形成。另一方面，erVP2蛋白已藉由發酵槽來進行大規模生產。本研究所探討的erVP2與erVP2H蛋白皆能組裝形成特定的結構，這是在以往的研究當中從未發現的現象，因此相信這兩種特定結構在雞隻的免疫過程中能誘發雞隻產生很強的免疫反應。

Abstract Gene encoding a structural protein (VP2) of infectious bursal disease virus (IBDV) was cloned and expressed using the host, Escherichia coli (E. coli), to investigate its capability of the formation of particulate structure. Although the previous attempt to produce unfused VP2 in E. coli was reported to be unsuccessful, in this report, the soluble E. coli-derived rVP2 (erVP2) proteins were found to form particles of approximately 20 nm in diameter. Those particles were partially purified employing CsCl density gradient ultracentrifugation, and confirmed by direct observation under the electron microscope. To facilitate the purification of the particles, the VP2 protein was incorporated a metal ion binding site (His)6 at its C-terminus. The chimeric erVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni+2). However, the chimeric erVP2H particles have a slightly smaller particulate size, approximately, 15 nm. The solubility of erVP2H is extremely low in the strain of BL21(DE3)pLysS and it was expressed with a limited amount in the strain of BL21(DE3)CodonPlus-RP. Besides, two C terminal deletion mutant proteins of VP2, 1167H and 1197H protein also failed to form particles. Thus, the erVP2 protein was selected to express in a laboratory-scale fermentor. In conclusion, the most significant finding in this work is that both of the expressed erVP2 and erVP2H proteins can form a particulate structure, which is not found previously and is believed to induce a strong immunological response in a vaccinated chicken.