Cloning, Expression, and Purification of the Recombinant Hemagglutinin of Human Influenza Virus H1n1 in the Eukaryotic Insect Cells Using Baculovirus Vector Publisher



Rashedi N¹ ; Taghizadeh M^{1, 2} ; Mohamadynejad P¹ ; Mahdavi M^{1, 3} ; Jalalirad R^{1, 4} Show Affiliations
Source: Journal of Isfahan Medical School Published:2020

Abstract

Background: The H1N1 influenza virus is a highly pathogenic virus that threatens human life. Vaccination is an effective way of preventing and controlling influenza. Production of recombinant hemagglutinin in the baculovirus expression system, in the insect eukaryotic cell substrate (Sf9), has been suggested as an effective strategy. Methods: The H1N1 influenza virus hemagglutinin gene sequence was prepared from National Center for Biotechnology Information (NCBI). After designing a specific primer, the sequence was provided using restriction digestion, cloned into pFastBacHTA plasmid, and transferred to the DH10Bac cell to produce a recombinant bacmid. After extracting the relevant plasmid, it was transfused into the insect cell; and after the expression of the protein by Sf9 cell, the presence of recombinant protein was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot methods. Findings: The hemagglutinin gene (654 bp) was cloned in pFastBacHTA plasmid using the two enzymes of BamHI and Xhol. Sf9 cell expressed a protein weighing approximately 60 kDa after receiving the recombinant bacmid protein. The extracted protein was identified and confirmed using SDS-PAGE and Western blot methods; and protein concentration was measured by Lowry method. Conclusion: The baculovirus system is useful for the production of proteins with complex structures. Generally, it can be concluded that this protein is highly expressed in insect cells. Due to the similarity of this system with the human system, it can be a suitable alternative for embryonic eggs in the future, and can be used in vaccination. © 2016 Isfahan University of Medical Sciences(IUMS). All rights reserved.