Branch of 48th Central Research Institute of Ministry of Defense of Russian Federation — Center of Virology, Sergiev Posad; Engelgart Institute of Molecular Biology, Moscow, Russia
Aim. To obtain human recombinant 70kDa heat shock protein (Hsp70) in baculovirus expression system and to study its antiviral activity. Materials and methods. Baculovirus expression system was used to obtain recombinant HSP70. Plasmid pFastBacHTb-Hsp70 containing sequence coding HSP70 gene with insertion of 6 histidine residues in protein reading frame was constructed. Competent cells MAX Efficiency DH 10 Bac were transfected with pFastBacHTb-Hsp70 plasmid with following extraction of recombinant bacmid Bac-Hsp70. In order to obtain baculovirus expressing HSP70, Sf-9 cells were transfected with Bac-Hsp70 bacmid. Hsp70 extraction and purification was performed with column metal-chelating affinity chromatography using Ni2+ ions. Protective efficacy of recombinant human HSP70 was estimated using model of Venezuelan equine encephalitis (VEE) in mice. Results. Recombinant bacmid Bac-Hsp70 was constructed based on Bac-to-Bac expression system. Baculovirus expressing human HSP70 have been produced after transfection of Sf-9 cells with Bac-Hsp70 bacmid. Cultivation of recombinant baculovirus in Sf-9 cells and application of metal-chelating affinity chromatography allowed to extract purified fraction of HSP70. Experiments on mice infected with VEE virus demonstrated significant protection from death after administration of HSP70 in dose 15 mcg/mice. Conclusion. Application of baculovirus expression system and insect cell line for accumulation of recombinant baculoviruses in combination with Ni2+-mediated metal-chelating affinity chromatography allowed to obtain highly purified human recombinant HSP70 with marked antiviral activity.
Zh. Mikrobiol. (Moscow), 2011, No. 1, P. 54—60