新型H7N9流感病毒重組核糖核蛋白與核蛋白鼠源單株抗體之開發

2013年3月，中國大陸爆發人類感染H7N9禽流感之疫情，同年4月，台灣也宣布第一起境外移入之H7N9病例。此為全球首次出現低致病性H7N9亞型病毒經基因重組引發人類感染，且某些病毒株也被驗出具有克流感之抗藥性。至2016年5月為止，全球已超過786個確定病例，死亡人數為307位，致死率高達39%。因此加強對H7N9禽流感病毒之基礎研究及研發快速篩檢與治療所需之抗體迫切重要。NP、PA、PB1、PB2及RNA組成核醣核蛋白複合體 (Ribonucleoprotein, RNP) 結構，扮演流感病毒遺傳物質RNA複製與轉錄的重要角色。其中NP會與病毒RNA纏繞，並且不僅能與病毒自身蛋白質有交互作用，在宿主體內更和多種蛋白質有結合現象，為一種多功能蛋白質，因此已成為目前研究抗流感病毒的標的之一。本論文的研究目標為表現與純化H7N9病毒NP、PA、PB1及PB2的重組蛋白質，並進行單株抗體的製備。本研究已成功利用昆蟲細胞穩定表現這四個蛋白質，並以親和層析法純化到NP，亦成功篩選出抗NP之單株抗體。其型別為IgG1 kappa，且該抗體具有可免疫沉澱NP的能力。此外，本研究發現PA、PB1及PB2之N端若含有鞘膜表面醣蛋白67 (envelope surface glycoprotein, GP67) 之signal peptide，能增加其蛋白質之表現量。然而，GP67-PA及GP67-PB1易形成不溶體，因此未能順利以層析法將其進行有效地純化出來。

關鍵字

新型H7N9流感病毒；核糖核蛋白；核蛋白；核蛋白單株抗體

並列摘要

In March 2013, the first human case infected by a novel avian influenza H7N9 virus subtype was reported in China. In April 2013, Taiwan CDC confirmed the first imported case of H7N9 infected patient from China. It is the first time that a low pathogenic avian influenza virus has led to human infection on account of genetic reassortment. Moreover, some isolated H7N9 viruses have shown Tamiflu drug-resistant. As of May, 2016, 786 documented human cases were confirmed by WHO and the mortality rate was as high as 39%. Therefore, it is urgently important to strengthen basic research of H7N9 and develop rapid laboratory diagnostics and antibodies. Influenza virus ribonucleoproteins (RNP), composed of NP, PA, PB1, PB2, and viral RNA, play important roles in viral gene transcription and replication. NP is a multifunction protein, which not only interacts with viral proteins but also interacts with host cell proteins. Therefore, NP is a potential antiviral target. The present study is aimed to express NP, PA, PB1 and PB2 recombinant proteins by for production of monoclonal antibodies. These four proteins have been stably generated in Sf21 insect cells and the NP recombinant proteins were purified by affinity chromatography. The anti-NP monoclonal antibody 2-4-7B showed great specificity and can be applied for NP immunoprecipition. In addition, this study found that the expression levels of PA, PB1 and PB2 proteins were increased by fusion of GP67 signal peptide at the protein N-terminus. However, PA, PB1 and PB2 were expressed as inclusion bodies, therefore the protein purification strategy should be further investigated.

並列關鍵字

Novel influenza A(H7N9) virus ； Ribonucleoproteins ； Nucleoprotein ； Anti-nucleoprotein monoclonal antibodies

參考文獻

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Chen, F., Li, J., Sun, B., Zhang, H., Zhang, R., Yuan, J., Ou, X., Ye, W., Chen, J., and Liu, Y. (2015). Isolation and characteristic analysis of a novel strain H7N9 of avian influenza virus A from a patient with influenza-like symptoms in China. International Journal of Infectious Diseases 33, 130-131.

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新型H7N9流感病毒重組核糖核蛋白與核蛋白鼠源單株抗體之開發

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