Cloning, Expression, and Purification of the Recombinant Hemagglutinin of Human Influenza Virus H1N1 in the Eukaryotic Insect Cells Using Baculovirus Vector

Document Type : Original Article (s)

Authors

1 PhD Student, Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

2 Invited Assistant Professor, Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran AND Assistant Professor, Department of Research and Development, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

3 Assistant Professor, Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

4 Invited Assistant Professor, Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran AND Assistant Professor, Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran

5 Invited Assistant Professor, Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran AND Assistant Professor, Production and Research Complex, Pasteur Institute of Iran, Karaj, Iran

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.

Keywords

References

  1. Munster VJ, Fouchier RA. Avian influenza virus: of virus and bird ecology. Vaccine 2009; 27(45): 6340-4.
  2. Alexander DJ. A review of avian influenza in different bird species. Vet Microbiol 2000; 74(1-2): 3-13.
  3. Knipe D, Howley P, Cohen J, Griffin D, Lamb R, Martin M, et al. Fields virology. Philadelphia, PA: Lippincott Williams and Wilkins; 2013.
  4. Roos R. WHO says H1N1 pandemic is over. Minneapolis, MN: Center for Infectious Disease Research and Policy Office of the Vice President for Research, University of Minnesota; 2010.
  5. Hussain M, Galvin HD, Haw TY, Nutsford AN, Husain M. Drug resistance in influenza A virus: the epidemiology and management. Infect Drug Resist 2017; 10: 121-34.
  6. Ligon BL. Avian influenza virus H5N1: A review of its history and information regarding its potential to cause the next pandemic. Semin Pediatr Infect Dis 2005; 16(4): 326-35.
  7. Hitchman RB, Possee RD, King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol 2009; 3(1): 46-54.
  8. Steel J. New strategies for the development of H5N1 subtype influenza vaccines: progress and challenges. BioDrugs 2011; 25(5): 285-98.
  9. Cox MM. Recombinant protein vaccines produced in insect cells. Vaccine 2012; 30(10): 1759-66.
  10. Berger I, Fitzgerald DJ, Richmond TJ. Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 2004; 22(12): 1583-7.
  11. Bright RA, Carter DM, Daniluk S, Toapanta FR, Ahmad A, Gavrilov V, et al. Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine 2007; 25(19): 3871-8.
  12. Kang SM, Pushko P, Bright RA, Smith G, Compans RW. Influenza virus-like particles as pandemic vaccines. Curr Top Microbiol Immunol 2009; 333: 269-89.
  13. Ludwig C, Wagner R. Virus-like particles-universal molecular toolboxes. Curr Opin Biotechnol 2007; 18(6): 537-45.
  14. Tinsley TW, Harrap KA. Viruses of Invertebrates. In: Fraenkel-Conrat H, editor. Newly Characterized Protist and Invertebrate Viruses. Boston, MA: Springer US; 1978. p. 1-101.
  15. Wang K, Holtz KM, Anderson K, Chubet R, Mahmoud W, Cox MMJ. Expression and purification of an influenza hemagglutinin-one step closer to a recombinant protein-based influenza vaccine. Vaccine 2006; 24(12): 2176-85.
  16. Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs. PLoS One 2008; 3(5): e2257.
  17. Hu YC, Luo YL, Ji WT, Chulu JL, Chang PC, Shieh H, et al. Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system. J Virol Methods 2006; 135(1): 43-8.
  18. Powers DC, Kilbourne ED, Johansson BE. Neuraminidase-specific antibody responses to inactivated influenza virus vaccine in young and elderly adults. Clin Diagn Lab Immunol 1996; 3(5): 511-6.
  19. Ahmad B, Li Z, Hanif Q, Hu Q, Wei X, Zhang L, et al. A Hybrid Peptide DEFB-TP5 Expressed in Methylotrophic Yeast Neutralizes LPS with Potent Anti-inflammatory Activities. Front Pharmacol 2020; 11: 461.
  20. Soleimanjahi H, Fotouhi F. Baculoviruses and insect cells as powerful tools for gene expression. Tehran, Iran: Jahad Daneshgahi; 2009. [In Persian].
  21. Luckow VA, Summers MD. Trends in the Development of Baculovirus Expression Vectors. Bio/Technology 1988; 6(1): 47-55.
  22. Kitts PA, Possee RD. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 1993; 14(5): 810-7.
Journal of Isfahan Medical School
Volume 38, Issue 572 - Serial Number 572
March and April 2020
Pages 260-266

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History

  • Receive Date: 18 April 2020
  • Revise Date: 02 November 2024
  • Accept Date: 06 April 2022

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