2014 ; 24(12):
|Author||Mohamed Rasheed Gadalla, Ayman Hany El-Deeb, Mohamed Maged Emara, Hussein Aly Hussein|
|Affiliation||Department of Virology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt|
|Title||Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter|
J. Microbiol. Biotechnol.2014 ; 24(12):
|Abstract||In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate
surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus
(AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1
promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully
cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into
DH10Bac competent cells, and the recombinant bacmid was generated via site-specific
transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9)
insect cells to construct the recombinant baculovirus and to induce expression of the HA
protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed
hemadsorption activity. Hemagglutination activity was also detected in both extra- and
intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by
reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was
observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE
analysis of the expressed protein revealed the presence of a visually distinguishable band of
~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis
confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of
H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The
expressed protein was displayed on the plasma membrane of infected insect cells under the
control of whispovirus ie-1 promoter by using the baculovirus expression vector system.|
|Keywords||Avian influenza, Hemagglutinin, Baculovirus, Whispovirus immediate early-1, Promoter|
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