Construction and identification of helper-dependent adenoviral vector encoding hemagglutinin protein of swin-origin influenza virus A H1N1 / 生物医学工程学杂志

Abstract: In order to investigate immune protection against swine-origin influenza virus (S-OIV) A H1N1, the helper-dependent adenovirus vector (HDAd) system was exploited to construct recombinant HDAd encoding hemagglutinin (HA). The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfected with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.

In vitro transgenic expression efficacy of a helper-dependent adenoviral vector encoding enhanced green fluorescent protein / 生物工程学报

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.