?d T Cells Provide Protective Function in Highly Pathogenic Avian H5N1 Influenza A Virus Infection

Dong, Peng; Ju, Xiangwu; Yan, Yiwu; Zhang, Siya; Cai, Menghua; Wang, Huaishan; Chen, Hui; Hu, Yu; Cui, Lianxian; Zhang, Jianmin; He, Wei

Monomeric and trimeric rHA proteins were expressed and purified using a baculovirus-insect cell system (Invitrogen, Thermo Fisher scientific, USA) as described previously (22) . First, the HA ectodomain DNA fragment of A/Anhui/1/2005 (H5N1, Accession No. DQ371928) and His tag were cloned into the transfer vector PacGP67b (BD Biosciences Pharmingen, USA) to allow the efficient secretion of monomeric rHA proteins. A new construct containing the bacteriophage T4 fibritin fold on trimerization sequence was generated to allow the efficient secretion of trimeric rHA proteins as previously reported (23) . Next, Sf9 cells were cotransfected with the monomeric or trimeric rHA transfer vectors and linearized baculovirus DNA (Invitrogen, Thermo Fisher scientific, USA) to produce recombinant baculoviruses containing the HA genes. Transfection and virus amplification were carried out according to the baculovirus expression system manual. The supernatant from infected Sf9 cells was collected and purified by Ni-NTA chromatography (GE Healthcare, USA) against the C-terminal His tag. Western blotting was performed using anti-His or anti-HA antibodies to confirm the rHA proteins. To demonstrate that the expressed HA fragments were properly folded, they were analyzed by a Viscotek 270 Max GPC/SEC system according to the manufacturer's instructions (Malvern, UK). Gel filtration chromatography was conducted using P4000 and P2500 columns (Malvern, UK) with a running buffer (pH 8.0) composed of 135 mm NaCl, 135 mm KCl, 1.5 mm KH2PO4, and 1.0 mm Na2HPO4·12H2O.