利用畢赤氏酵母菌醱酵生產台灣GII.4 2006b型諾羅病毒類病毒顆粒及小型P粒子之純化與性質研究

Abstract

諾羅病毒為全球流行之非細菌性腸胃道致病菌。由於缺乏細胞株生產減毒或去活的諾羅病毒疫苗，諾羅病毒疫苗主要以類病毒顆粒為基礎發展。諾羅病毒衣殼蛋白質VP1之突出區(P (protruding) domain)，會構成由12個雙體化P蛋白質所組成之P粒子(P particle)，其抗原形態與病毒衣殼蛋白質VP1相同。因突出區具有三個多出來的環狀結構，能嵌入外來抗原，先前已做為外來抗原呈現平台。本研究欲使用分離自台灣本土株，GII.4型諾羅病毒之突出區作為P蛋白質多抗原呈現平台之基礎。首先在表現部分，利用不具有內毒素疑慮、培養成本低廉且易於放大培養之畢赤氏酵母菌Pichia pastoris，經醱酵槽培養，胞內P蛋白質產量可達220 mg/L。其次，在純化策略方面，利用P蛋白質本身的電性及原態具有的組胺酸，本研究發展出二套無標籤純化策略：(1)利用宿主蛋白質與P蛋白質對管柱的吸附落差，P蛋白質可經由離子交換管柱及疏水性管柱純化得到，但最終回收後產量僅有2.5%。為進一步提升產量，(2)利用親和層析管柱(His-Trap)能與原態暴露在外之組胺酸吸附的特性，P蛋白質可利用親和層析管柱及離子交換管柱純化，回收率為28.1%，純度可達82.1%。此外，藉由VP1蛋白質原態所具有的組胺酸，及VP1能組成顆粒之特性，原態之VP1顆粒亦能藉由親和層析管柱及膠體層析管柱純化，回收率為20%，純度可超過90%。純化出之VP1蛋白質經粒徑分析儀及穿透式電子顯微鏡檢測，VP1能成功組成具有生物功能之粒子，能與成人唾液之HBGA (human histo-blood group antigen)結合。與帶有組胺酸標籤(His-tag)之P蛋白質(P-His)相似，純化出之原態P蛋白質，經粒徑分析儀測定及穿透式電子顯微鏡觀測，二種P粒子皆呈現小型P粒子(small P particle)之三角形及四角形形態，顯示以酵母菌表現之台灣流行株GII.4型諾羅病毒P蛋白質，能構成由6個雙體化P蛋白質組成之小型P粒子。前人文獻指出，小型P粒子僅出現在C端突變之P蛋白質。而本研究之P蛋白質未經末端修飾即呈現小型P粒子，經與其它GII.4菌株序列比對，發現其中四個胺基酸對P粒子的形成可能扮演關鍵角色。當以綠色螢光蛋白質模擬外來抗原，呈現於P蛋白質的表面頂端，結果發現嵌合型P蛋白質不但能成功組成嵌合小型P粒子，亦能發出綠色螢光，顯示台灣分離株諾羅病毒P蛋白質能做為外來抗原呈現平台。本研究為首篇提供諾羅病毒P蛋白質酵母菌大量生產、無標籤簡易純化及確立小型P粒子亦能作為外來抗原呈現平台之研究。

Norovirus (NoV) is one of the leading causes of acute nonbacterial gastroenteritis outbreaks worldwide. Due to the lack of a reliable and efficient cell culture system for producing inactivated or attenuated whole NoV vaccines, the development of NoV vaccines relies largely on virus-like particles (VLPs) formed by the major capsid protein VP1 or subviral particles formed by the exterior protrusion (P) domain of VP1. The P particle is composed of 12 P dimers and revealed the same antigenic types as VLP. Additionally, due to the presence of three outermost surface loops in the P domain, the P particle could serve as a platform for carrying foreign antigens. The goal of this study is to provide the fundamental understanding of different constructs of P particles formed by the P domain of NoV strain GII.4 isolated from Taiwan to establish the multiple antigen presentation platform. The P domain was expressed in Pichia pastoris, a well-known expression system with several advantages including high cell density fermentation at low cost and non-risk of endotoxin. The production of NoV P protein reached 220 mg/L as a soluble form in fermentation cultures. The overexpression P protein provided the cornerstone for the development of tag-free P protein purification schemes. For purification of tag-free P protein, based on the charge and the surface histidine in native NoV, two purification schemes were developed: (1) The host cell proteins and the target P protein were separated by the difference of the binding strength to the chromatography column. Using anion-exchange and hydrophobic interaction chromatography purification schemes, NoV P protein with high purity was obtained. However, the P protein recovery was 2.5%. (2) Using the HisTrap affinity column and anion-exchange column, the native NoV P protein was purified, and recovery and purity were 28.1% and 82.1%, respectively. Besides, the NoV major capsid protein VP1 was also purified using the HisTrap affinity column and gel filtration column. The purity of NoV VP1 protein was over 90% and the recovery was 20%. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis of the purified NoV VP1 revealed that VP1 proteins were self-assembled into particles, and these particles remained HBGA binding ability as evidenced by saliva binding assay. Similar to P-His protein, the P protein also formed biologically functional small P particle composed by six of P dimer. The purified P-His and P protein, analyzed by TEM and DLS, were also revealed triangle-, square- and ring-shaped. Previous studies showed that the small P particles were only found with C-terminus modification. Without terminal modification, small P particles were formed in this study. The amino acid sequence analysis showed only four different amino acids between the P domain in this study and other investigated GII.4 strains, suggesting that these amino acids might play an important role in P particle formation. To extend the application of the small P particle, the green fluorescent protein was used to mimic the foreign antigen and inserted into NoV P loop2 distal end. From gel filtration and fluorescent microscopy, the chimera small P proteins were self-assembly into chimera small P particles and showed green fluorescence. It was indicated that these Taiwan native small P particle can be an antigen presentation platform. This study was the first report of NoV P protein covering overexpression in P. pastoris, easy handling tag-free purification schemes, and formation of small P particles without terminal modification as foreign antigen displaying platform.