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

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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

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

盧德釗 , Masters　　Advisor：張世宗　　

繁體中文

新型H7N9流感病毒；基質蛋白；非結構性蛋白；桿狀病毒表現系統； Novel H7N9 influenza virus ； Matrix protein ； Nonstructural protein ； Baculovirus expression system

Abstract │ Reference (38) │ Altmetrics

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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.

Reference ( 38 ) 〈TOP〉

Ayllon, J., Domingues, P., Rajsbaum, R., Miorin, L., Schmolke, M., Hale, B.G., and Garcia-Sastre, A. (2014). A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence. J Virol 88, 12146-12151.
連結：
Belser, J.A., Gustin, K.M., Pearce, M.B., Maines, T.R., Zeng, H., Pappas, C., Sun, X., Carney, P.J., Villanueva, J.M., Stevens, J., et al. (2013). Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature 501, 556-559.
連結：
Belshe, R.B. (2005). The origins of pandemic influenza--lessons from the 1918 virus. N Engl J Med 353, 2209-2211.
連結：
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.
連結：
Chan, P.K. (2002). Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in 1997. Clin Infect Dis 34 Suppl 2, S58-64.
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