Construction of Recombinant Bacmid DNA Encoding Influenza Virus A (H۱N۱) Hemagglutinin Gene

Objective: Influenza virus A (H۱N۱) 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 HA۱ genes to generate recombinant baculovirus shuttle vectors (bacmid) in order to produce recombinant proteins in insect cells. Methods: Human influenza virus A/New Caledonia ۹۹/۲۰/(H۱N۱) was propagated in MDCK cell culture. Total viral RNA was extracted using easy-red solution. The full-length HA genome and HA۱ 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 DH۱۰Bac™ 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/M۱۳ or gene-specific primers. Conclusion: In this study, we have successfully constructed recombinant Bacmid DNA that encoded the full-length HA genome and its HA۱ subunit. We intend to transfect sf۹ insect cells with these constructs to generate recombinant baculovirus and produce large amounts of desired proteins for future studies.

کلیدواژه ها:

Recombinant DNA ، Baculovirus ، Hemagglutinin ، Subunit Vaccine ، DNA نوترکیب ، باکیولوویروس ، هماگلوتینین ، واکسن زیرواحدی