傳染性華氏囊病毒結構蛋白VP2之C端區域對形成似病毒顆粒及免疫力之影響

Abstract

為了了解傳染性華氏囊病毒結構蛋白VP2的C端如何影響VP2蛋白形成virus-like particles，本研究利用昆蟲細胞/桿狀病毒表現系統表現3個不同C端長度之VP2突變蛋白，即成熟型VP2ΔC11、C端截切41個胺基酸的VP2ΔC41及C端延伸25個胺基酸的重組蛋白VPXΔC46。這三種蛋白的C端皆融合6個His，但只有VP2ΔC11能有效以利用固定化金屬親和性色層層析法（Immobilized metal-ion affinity chromatography, IMAC）純化，VPXΔC46之純化效率則大為減弱，而VP2ΔC41幾乎無法以IMAC純化，必須藉由氯化銫密度梯度離心、HPLC進行純化。 經由純化所得之蛋白質，以穿透式電子顯微鏡觀察，發現VP2ΔC11可以組裝成粒徑大小約20 nm的似病毒顆粒，VP2ΔC41則無法形成，表示傳染性華氏囊病毒結構蛋白VP2的A-411到K-437的胺基酸在病毒顆粒的組裝中扮演重要角色，而K-437到A-441會影響蛋白組裝，VPXΔC46形成結構鬆散的構型。 由免疫試驗結果得知經由IMAC純化所得之重組結構蛋白VP2ΔC11具有引發雞隻免疫保護之能力，所以利用500 ml磁攪拌培養瓶生產重組蛋白，並比較不同multiplicity of infection (MOI)對蛋白產量之影響，當MOI為 1時感染細胞，蛋白產量較高。以昆蟲細胞生產重組蛋白VP2ΔC11並不完全形成似病毒顆粒，可分為似病毒顆粒和非似病毒顆粒兩種型態，經由雞隻免疫試驗，發現VP2ΔC11似病毒顆粒可誘發雞隻產生中和性抗體，保護雞隻不受傳染性華氏囊病毒感染，而VP2ΔC41則無法引起中和性抗體。同時亦將VP2ΔC11蛋白似病毒顆粒進行蛋內注射，初步認為蛋內注射的實驗並不能引發雞隻產生抗傳染性華氏囊病毒之抗體。

VP2 is the major structural protein of infectious bursal disease virus(IBDV)and the host-protective antigen which is responsible for induction of virus-neutralizing antibodies that protect chickens from IBDV infection. In this study, three mutant proteins of VP2, including VP2ΔC11, VP2ΔC41 and VPXΔC46 were expressed in baculovirus expression system to evaluate their self-assembly and protective capability. The result showed that the C terminal truncated protein, VP2ΔC11, assembled into particle structure in a diameter of 20 nm, whereas VP2ΔC41 and VPXΔC46 could not form particle structure. The results suggest that C-terminal regions of VP2, amino acid residues ranging from 411 to 437, are critical for the particulate formation. The protective efficacy of the purified VP2ΔC11 particles and VP2ΔC41 were evaluated using chicken protection assay. Our result indicated that the VP2ΔC11 particles can protect chicken from infectious bursal disease virus infection, but VP2ΔC41 not. However in ovo vaccination with VP2ΔC11 could not prevent young chicken from the infection of infectious bursal disease virus.