利用桿狀病毒表現系統表現H5N1之單鏈可變區片段抗體

Expression of H5N1 Single-chain Variable Fragment Antibodies by using Baculovirus Expression System

吳郁蓉(Yu-Rong Wu)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6840/cycu201400771

摘要

A型流感病毒H5N1又被稱為高致病性流感病毒（高致病性禽流感），主要感染家禽和鳥類，會對鳥類造成嚴重症狀，甚至死亡。在1997年香港爆發人類感染禽流感之病例。根據世界衛生組織統計，從2003年至2013年6月H5N1病毒已造成375人死亡和高達60％的死亡率，相較於其他A型流感病毒，死亡率高出10-100倍。因此，禽流感病毒已成為全球都必須關注的全球性公共健康問題。在此研究中，我們利用昆蟲桿狀病毒表達載體系統（BEVS）表現H5N1單鏈可變片段（scFv）形式的抗體，單鏈可變片段（scFv）形式的抗體主要包含免疫球蛋白抗體的重鏈（heavy chain）和輕鏈（light chain）的可變區（variable regions），兩段基因之間利用10至25個氨基酸的短胜肽做連接形成融合蛋白。已有研究指出scFv可用於診斷，治療和研究。另一方面，在實驗室之前的研究顯示，共同表現的分子伴侶β-突觸核蛋白（β-synuclein）或真核轉譯起始因子eIF4E可以在BEVS中提高分泌的蛋白質生產以及提高細胞的存活率。因此，利用含有eIF4E與β-突觸核蛋白的四效表現載體表現H5N1的scFv抗體以提高在昆蟲桿狀病毒表達載體系統（BEVS）的產量和延長細胞存活率的可能性。我們成功的利用昆蟲桿狀病毒表達載體系統表現H5N1的scFv抗體，並且透過western blot 進一步的分析。此外，我們驗證了共表現β-突觸核蛋白和真核轉譯起始因子eIF4E可以延長細胞存活率，並且利用Ni-NTA column成功純化到H5N1的scFv抗體，在未來希望透過競爭型ELISA或中和能力的相關實驗進一步證實其抗體之功能，並且我們希望能應用在診斷或治療H5N1的感染。

關鍵字

單鏈可變區片段抗體 ； H5N1 ； 桿狀病毒表現系統

並列摘要

Influenza A H5N1 virus also known as highly pathogenic influenza viruses（HPAIV）are mainly spread among poultry and birds with severe symptoms or even death. The first case of human infection H5N1 from poultry is reported in Hong Kong in 1997. According to World Health Organization statistics , H5N1 have caused 375 deaths and up to 60 percent mortality rate from 2003 to June 2013. Comparing to other influenza A virus, mortality rate caused by H5N1 is out of 10-100 fold. Therefore, the influenza virus is a global public health problem which requires a high degree of attention. In this study, we express the single-chain variable fragment（scFv）generated from coding gene of H5N1 monoclonal antibody by baculovirus expression vector system（BEVS）. The scFv is a fusion protein of the variable regions of heavy chain（VH）and light chains（VL）of immunoglobulins, which connected with a short linker peptide of 10 to 25 amino acids. ScFv can be useful for diagnosis, therapy and research. In addition, our previous studies indicated that co-expression of the chaperon β-synuclein（β-syn）or a translation initiation factor eIF4E can enhance secreted protein productions in BEVS. Thus, a further combination of eIF4E and b-syn with scFv raise the possibility to increase the production of scFv in BEVS. We planed to express secreted scfv against HA protein of H5N1 in BEVS. After successfully cloned the scfv into expression vectors and generated the purified recombinant viruses, we could detect the scFv in infected cell lysate and medium by western blot analysis. Furthermore, we found that β-syn and eIF4E could enhance scFv protein secretion compared to the control. We can use BEVS to express and purify H5N1 scFv antibodies successfully by Ni-NTA column and we also validated that co-expression of β-syn and eIF4E together can prolongs cell viability. In the future, the function of purified ScFv such as competitive ELISA or neutralizing ability need to be further confirmed. We hope that it could expand the application in diagnostic or therapeutic for H5N1 infection.

並列關鍵字

Baculovirus expression system ； H5N1 ； Single-chain variable fragment（scFv）

參考文獻

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