The wt NA and mutant NA (I223R/H275Y) genes derived from A/H1N1 2009 pandemic influenza virus were amplified by PCR. The PCR products were cloned into the
pAcGP67A vector (BD Biosciences) and transfected into the Sf9 insect cell line to produce recombinant proteins. The culture medium was collected, clarified by centrifugation at 110 ×
g, and subjected to further viral propagation. The cultured Sf9 cells were inoculated with recombinant baculovirus at a multiplicity of infection of 10 and incubated at 27 °C for 96 h. The cells were collected after centrifugation, and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 16,000 ×
g for 15 min at 4 °C. After the cell lysate was sonicated to reduce its viscosity, the cell debris was removed by centrifugation for 1 h at 16,000 ×
g. The soluble protein from the cell supernatant was applied to
Ni-nitrilotriacetic acid agarose resin (Qiagen), washed, and eluted with buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 8.0). The purified proteins were dialyzed against phosphate-buffered saline (PBS) and further purified by
Q-Sepharose anion-exchange chromatography (GE Healthcare).
Guk K., Kim H., Lee M., Choi Y.A., Hwang S.G., Han G., Kim H.N., Kim H., Park H., Yong D., Kang T., Lim E.K, & Jung J. (2020). Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus. Nature Communications, 11, 3418.