Hossainzadeh, Samaneh, Fotouhi, Fatemeh, Farahmand, Behrokh, Saleh, Maryam, Yousefi, Atena, Heydarchi, Behnaz, Tavasoti Kheiri, Masoumeh (2012). Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H1N1) Hemagglutinin Gene. , 14(4), 39-49.
Samaneh Hossainzadeh; Fatemeh Fotouhi; Behrokh Farahmand; Maryam Saleh; Atena Yousefi; Behnaz Heydarchi; Masoumeh Tavasoti Kheiri. "Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H1N1) Hemagglutinin Gene". , 14, 4, 2012, 39-49.
Hossainzadeh, Samaneh, Fotouhi, Fatemeh, Farahmand, Behrokh, Saleh, Maryam, Yousefi, Atena, Heydarchi, Behnaz, Tavasoti Kheiri, Masoumeh (2012). 'Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H1N1) Hemagglutinin Gene', , 14(4), pp. 39-49.
Hossainzadeh, Samaneh, Fotouhi, Fatemeh, Farahmand, Behrokh, Saleh, Maryam, Yousefi, Atena, Heydarchi, Behnaz, Tavasoti Kheiri, Masoumeh Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H1N1) Hemagglutinin Gene. , 2012; 14(4): 39-49.

Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H1N1) Hemagglutinin Gene

Pathobiology Reserach
Article 4, Volume 14, Issue 4, 2012, Pages 39-49    PDF (909.96 K)
Authors
Samaneh Hossainzadeh1; Fatemeh Fotouhi* 2; Behrokh Farahmand2; Maryam Saleh2; Atena Yousefi1; Behnaz Heydarchi2; Masoumeh Tavasoti Kheiri3
1Department of Biology, Faculty of Basic Sciences, Science and Research branch of Islamic Azad University, Tehran, Iran
2Influenza Research Lab, Pasteur Institute of Iran, Tehran, Iran
3Influenza Lab research, Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
Abstract
Objective: Influenza virus A (H1N1) is an important subtype of the influenza respiratory viruses, which has important worldwide implications. Hemagglutinin (HA), an important viral antigen, is responsible for binding to human cell receptors leading to an onset of the disease process. Considering the critical role of viral attachment, this study focuses on the extraction and cloning of HA and its large subunit HA1 genes to generate recombinant baculovirus shuttle vectors (bacmid) in order to produce recombinant proteins in insect cells.
Methods: Human influenza virus A/New Caledonia 99/20/(H1N1) was propagated in MDCK cell culture. Total viral RNA was extracted using easy-red solution. The full-length HA genome and HA1 fragment were amplified by RT- PCR using specific primers, cloned into a pGEM®-TEasy vector, and then subcloned into a pFastBac HT plasmid. Finally, recombinant bacmids that contained the genes of interest were produced in E. coli DH10Bac™ cells.
Results: Expected PCR products of HA genes were evaluated through gel electrophoresis and restriction enzyme analysis. Recombinant pGEM®-TEasy vectors and pFastBac HT donor plasmids were confirmed by PCR, digestion, and sequencing. Construction of recombinant bacmid DNA was verified by using blue-white colony screening, overnight electrophoresis, and PCR analysis that used either pUC/M13 or gene-specific primers.
Conclusion: In this study, we have successfully constructed recombinant Bacmid DNA that encoded the full-length HA genome and its HA1 subunit. We intend to transfect sf9 insect cells with these constructs to generate recombinant baculovirus and produce large amounts of desired proteins for future studies.
Keywords
Recombinant DNA; Baculovirus; Hemagglutinin; Subunit Vaccine
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