Expression and Purification of the Novel H7N9 Influenza A Virus Recombinant Matrix Proteins and Nonstructural Proteins

• Main Authors: De-Chao Lu, 盧德釗
• Other Authors: 張世宗
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/12543633400412377493

碩士 === 國立臺灣大學 === 生化科技學系 === 104 === 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.