表現與純化新型H7N9流感病毒M1、NS1和NS2重組蛋白及製備鼠源單株抗體

Abstract

2013年3月中國爆發全球首次由低致病性H7N9禽流感病毒經基因重組而引發高致病性的人類感染。臺灣也於同年4月宣布首例之境外移入病例。根據世界衛生組織調查，截至2016年6月，確診病例已超過700例，死亡率更高達近四成。研究指出此H7N9的基質蛋白M1已產生N30D及T215A的突變，會使病毒的病原性增強；與形成核醣核蛋白複合體有關的非結構性蛋白NS1已產生P42S的突變，會使病毒的病原性增強，顯示新型H7N9禽流感病毒對哺乳類宿主已開始具有適應性，且M1、NS1及NS2等蛋白在病毒感染宿主及複製時皆有其重要的功能，因此本研究的目標為表現並純化新型H7N9流感病毒的M1、NS1、及NS2的重組蛋白以進行專一性抗體之製備。本研究已成功表現出M1、NS1及NS2的重組蛋白質，並藉由親和層析法分別進行純化且完成量產，並將M1、NS1及NS2 分別免疫BALB/c小鼠後產生多株抗體。利用單株抗體的篩選及純化，已成功得到抗NS1的單株抗體。ELISA及免疫染色法的結果顯示此抗NS1單株抗體具有高度專一性及效價，並可利用此抗體進行NS1之免疫沉澱，顯示此抗體與抗原間具有高度結合能力。

Since March 2013, more than 700 laboratory-confirmed human H7N9 influenza A virus infection cases have been reported by WHO as stated in June 2016, with a case fatality rate of more than 39%. Based on genetic analysis, this avian influenza A virus H7N9 shows some extent adaptation to mammalian hosts. It is reported that the novel H7N9 M1 has N30D and T215A mutations and NS1 has P42S mutation, which lead to increased virulence in mice. Therefore, it is urgently important to strengthen basic research of H7N9 and develop rapid laboratory diagnostics and antibodies. This study focused on production of the novel H7N9 influenza virus M1, NS1 and NS2 recombinant proteins for generation of antibodies. M1, NS1 and NS2 recombinant proteins were purified by affinity chromatography, and the purified M1, NS1 and NS2 proteins were used to immunize BALB/c mice for producing polyclonal antibodies. The anti-NS1 antibodies were screened by ELISA and further analyzed by western blotting. The anti-NS1 mAbs were purified, and the specificity and binding capacity toward H7N9 NS1 were analyzed by western blotting and immunoprecipitation.