Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system

Journal of Virological Methods
Yu C HuHung J Liu

Abstract

Baculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10B(AC)Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was usef...Continue Reading

References

May 23, 1998·The Biochemical Journal·C Freeman, C R Parish
Jan 19, 2002·Journal of Virology·Christoph ScholtissekRobert G Webster
Apr 9, 2005·Journal of Clinical Microbiology·Xiaojuan XuHuanchun Chen
Apr 30, 2005·Analytical Chemistry·Ruiqing Qiu, Fred E Regnier
Jul 8, 2005·Journal of Clinical Microbiology·Majid EshaghiKhatijah Yusoff

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Citations

Mar 22, 2007·Molecular Therapy : the Journal of the American Society of Gene Therapy·Ding-Gang YangYu-Chen Hu
Jun 19, 2012·Bioengineered·Hsin-Yu LuHung-Jen Liu
Aug 31, 2007·Journal of Virological Methods·Chi Y WangHung J Liu
May 10, 2013·Proceedings. Biological Sciences·Thomas E CurrieRuth Mace

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