新型H7N9流感病毒重組基質蛋白與非結構性蛋白之表現與純化

Abstract

在2013年3月中國大陸爆發人感染H7N9禽流感病毒之疫情，此為全球首次低致病性H7N9禽流感病毒經基因重組引發高致病性人類感染。臺灣在同年4月出現首例境外移入病例，在其體內驗出具有克流感抗藥性的H7N9病毒。目前全球超過600位的確定病例，死亡率高達40%。根據基因序列分析，發現此禽流感病毒對哺乳類宿主具有適應性。因此研發快速檢驗與治療所需之抗體非常重要。本論文研究目標為利用桿狀病毒表現系統，進行新型H7N9流感病毒的基質蛋白M1、M2與非結構性蛋白NS1、NS2重組蛋白質的生產，以提供基礎科學研究、抗體與疫苗研發之需求，有助於未來運用在流感病毒之監測與實驗診斷。本研究已將M1、M2、NS1與NS2基因分別選殖至pFastBac HT A載體，再轉型至E. coli DH10Bac中，以建立重組Bacmid載體。經轉染至Sf21昆蟲細胞後，已成功表現出H7N9流感病毒之M1、M2、NS1與NS2重組蛋白質。進一步將取得之重組桿狀病毒液進行效價測定，並測試M1、NS1與M2之最佳表現條件，得出以5 MOI的病毒液，感染Sf21之懸浮細胞5天的表現條件為最佳。將M1與NS1藉由親和層析法進行純化，並以質譜儀確認純化後的M1與NS1重組蛋白質的序列與預測之序列相符。綜合上述結果，本研究已成功利用桿狀病毒與昆蟲細胞表現與量產新型H7N9流感病毒M1與NS1的重組蛋白質，並將其分別免疫BALB/c小鼠產生多株抗體。

In March 2013, a new avian influenza A(H7N9) virus was reported to have infected humans for the first time in China. The novel H7N9 virus appears to be low pathogenic in birds but highly pathogenic in humans. In April 2013, Taiwan CDC reported the first imported case of H7N9 infection from China, and the H7N9 virus variants isolated from this patient performs Tamiflu resistance. A total of 693 laboratory-confirmed H7N9 influenza infections with a mortality of 40% was reported by WHO in December 2015. Based on genetic analysis, this avian influenza A virus H7N9 shows some extent adaptation to mammalian hosts. Therefore, it is very important to develop rapid laboratory diagnostics, antibodies and vaccines for better pandemic preparedness. This study focused on production of recombinant matrix proteins M1, M2 and nonstructural proteins NS1, NS2 of the novel H7N9 influenza virus by using baculovirus expression system for generation of antibodies, laboratory diagnosis and virological surveillance in the future. The gene encoding M1, M2, NS1, or NS2 has been cloned into pFastBac HT A respectively, and then transformed to E. coli DH10Bac competent cells for construction of recombinant bacmids, which were subsequently tranfected to Sf21 insect cells for expressing H7N9 M1, M2, NS1 and NS2 recombinant proteins. A viral plaque assay was applied for measuring the titer of the baculoviral stock. The optimal expression conditions of M1, NS1 and M2 were determined as using 5 MOI to infect cells for 5 days. Recombinant M1 and NS1 proteins with a 6xHis tag were purified by affinity chromatography, and the identity was confirmed by mass spectrometry. The high-level production of the recombinant H7N9 M1 and NS1 proteins by baculovirus expression system was successful in the present study. The purified H7N9 M1 or NS1 proteins were used to immunize the BALB/c mice to produce polyclonal antibodies.