tetano
Editor, Senior Moderator
J Microbiol Biotechnol. 2014 Aug 11. [Epub ahead of print]
Insect cell surface expression of Hemagglutinin (HA) of Egyptian H5N1 avian influenza virus under transcriptional control of whispovirus immediate early-1 Promoter.
Gadalla MR1, El-Deeb AH, Emara MM, Hussein HA.
Author information
Abstract
In the present study whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system (BEVS). The (HA) gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into 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 the expression of HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity, Hemagglutination (HA) activity was also detected in both extra- and intra-cellular recombinant HA. Both 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 ~63kDa 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 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.
PMID:
25112319
[PubMed - as supplied by publisher]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/25112319
Insect cell surface expression of Hemagglutinin (HA) of Egyptian H5N1 avian influenza virus under transcriptional control of whispovirus immediate early-1 Promoter.
Gadalla MR1, El-Deeb AH, Emara MM, Hussein HA.
Author information
Abstract
In the present study whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system (BEVS). The (HA) gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into 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 the expression of HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity, Hemagglutination (HA) activity was also detected in both extra- and intra-cellular recombinant HA. Both 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 ~63kDa 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 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.
PMID:
25112319
[PubMed - as supplied by publisher]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/25112319