腸病毒七十一型類顆粒之純化及其免疫活性之評估

Purification and immunogenicity of enterovirus-71 virus-like particles

王惠儀(Hui-Yi Wang)

指導教授 ： 陳俊任

國立臺灣大學/生命科學院/生化科技學系/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10406

摘要

腸病毒七十一型(enterovirus-71; EV-71)感染後，會造成手足口症，且易引起神經性併發症及死亡，在五歲以下的幼兒中有極高的致死率。於 1998 年時，在臺灣即爆發首次的大流行，然而目前卻沒有EV-71 的疫苗藥物供給預防。本研究之目的，在探討以嗜甲醇酵母菌表現系統生產的 EV-71 類病毒顆粒之最佳純化條件，並評估以其作為抗原之免疫效果。首先以一次非連續蔗糖梯度超高速離心純化 EV-71 類病毒顆粒，並將之進行動物免疫，然而成效不佳，推測可能肇因於類病毒顆粒之純度不高。因此進一步比較不同純化方法，包含蔗糖梯度與氯化銫梯度超高速離心，對類病毒顆粒之純度及產量的影響。在不同方式純化後，分別以SDS-PAGE與Western-blot分析類病毒顆粒之純度，並以穿透式電子顯微鏡觀察類病毒顆粒的產生。比較不同方法之純化結果，發現以二次非連續蔗糖梯度超高速離心純化方式，可得到最高的類病毒顆粒純度 (約 45%) 與總產量 (約 0.88 mg/L)。另外，在氯化銫梯度超高速離心後，得知EV-71類病毒顆粒的密度約為 1.22 g/ml。以二次非連續蔗糖梯度純化之類病毒顆粒作為抗原於小鼠進行免疫後，可有效引起體液免疫反應，其誘發之EV-71 專一性 IgG 抗體量，與以熱去活化 (heat-inactivated) EV-71為抗原時相當。綜合本研究之結果，顯示以酵母菌生產之EV-71類病毒顆粒具有發展成為疫苗之潛力。

關鍵字

腸病毒七十一型 ； 類病毒顆粒 ； 疫苗

並列摘要

Enterovirus-71 (EV-71) infection causes hand, foot and mouth disease (HFMD) and is prone to be complicated by severe neurological diseases, causing significant morbidity and mortality in children under 5 years of age. In 1998, EV-71 resulted in a large outbreak in Taiwan; however, there is no effective vaccine for EV-71 to date. In this study, we investigated the optimal method for purifying EV-71 virus-like particles (VLPs) expressed in Pichia pastoris, and evaluated the immunogenicity of purified VLPs in mice. We first purified EV-71 VLPs by a single discontinuous sucrose gradient ultracentrifugation, and found that the purified VLPs did not stimulate a strong immune response in mice, which was probably due to the presence of impurity in VLP samples. Therefore, we compared the purification of VLPs using different methods, including discontinuous sucrose gradient and CsCl gradient ultracentrifugations, and analyzed the purity and yield of VLPs by SDS-PAGE and Western blot. The results indicated that the highest VLP purity (about 45%) and yield (0.88 mg/L) could be obtained using double discontinuous sucrose gradient ultracentrifugation. The density of EV-71 VLP was determined to be about 1.22 g/ml by CsCl density gradient. Immunization of mice with the VLPs purified by double sucrose density gradient led to the production of anti-EV-71 IgG antibody, with a titer similar to that produced when heat-inactivated EV-71 was used as the immunogen. Taken together, our results indicate that the EV-71 VLPs produced in yeast have the potential to be developed as a vaccine candidate.

並列關鍵字

Key words：enterovirus-71 (EV-71) ； virus-like particles (VLPs) ； vaccine

參考文獻

1. Bible, J. M., P. Pantelidis, P. K. Chan, and C. Y. Tong. 2007. Genetic evolution of enterovirus 71: epidemiological and pathological implications. Reviews in medical virology 17: 371-379.

2. Weng, K. F., L. L. Chen, P. N. Huang, and S. R. Shih. 2010. Neural pathogenesis of enterovirus 71 infection. Microbes and infection / Institut Pasteur 12: 505-510.

3. Solomon, T., P. Lewthwaite, D. Perera, M. J. Cardosa, P. McMinn, and M. H. Ooi. 2010. Virology, epidemiology, pathogenesis, and control of enterovirus 71. The Lancet infectious diseases 10: 778-790.

4. Brown, B. A., M. S. Oberste, J. P. Alexander, Jr., M. L. Kennett, and M. A. Pallansch. 1999. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. Journal of virology 73: 9969-9975.

5. Koroleva, G. A., L. A. Gracheva, and M. K. Voroshilova. 1978. [Isolation of type 71 enterovirus from patients with a poliomyelitis-like disease during an outbreak in Bulgaria]. Voprosy virusologii: 611-618.