CONSTRUCTION OF RECOMBINANT BACMID DNA ENCODING INFLUENZA VIRUS A (H1N1) HEMAGGLUTININ GENE

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HOSSEINZADEH SAMANEH; FOTOUHI FATEMEH; FARAHMAND BEHROKH; SALEH MARYAM; YOUSEFI ATENA; HEYDARCHI BEHNAZ; TAVASOTI KHEIRI MASOUMAH

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Journal: Pathobiology Research Year:2012 | Volume:14 | Issue:4 Page(s): 38-47

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Title

CONSTRUCTION OF RECOMBINANT BACMID DNA ENCODING INFLUENZA VIRUS A (H1N1) HEMAGGLUTININ GENE

Author(s)

Keywords

RECOMBINANT DNAQ2

BACULOVIRUSQ2

HEMAGGLUTININQ3

SUBUNIT VACCINEQ2

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 DH10BacTM 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.

Cites

PURIFICATION OF INFLUENZA VIRUS A (H1N1) RECOMBINANT HEMAGGLUTININ (HA1) AND POLYCLONAL ANTIBODY PRODUCTION

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APA: Copy

HOSSEINZADEH, SAMANEH, FOTOUHI, FATEMEH, FARAHMAND, BEHROKH, SALEH, MARYAM, YOUSEFI, ATENA, HEYDARCHI, BEHNAZ, & TAVASOTI KHEIRI, MASOUMAH. (2012). CONSTRUCTION OF RECOMBINANT BACMID DNA ENCODING INFLUENZA VIRUS A (H1N1) HEMAGGLUTININ GENE. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), 14(4), 38-47. SID. https://sid.ir/paper/80953/en

Vancouver: Copy

HOSSEINZADEH SAMANEH, FOTOUHI FATEMEH, FARAHMAND BEHROKH, SALEH MARYAM, YOUSEFI ATENA, HEYDARCHI BEHNAZ, TAVASOTI KHEIRI MASOUMAH. CONSTRUCTION OF RECOMBINANT BACMID DNA ENCODING INFLUENZA VIRUS A (H1N1) HEMAGGLUTININ GENE. PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES)[Internet]. 2012;14(4):38-47. Available from: https://sid.ir/paper/80953/en

IEEE: Copy

SAMANEH HOSSEINZADEH, FATEMEH FOTOUHI, BEHROKH FARAHMAND, MARYAM SALEH, ATENA YOUSEFI, BEHNAZ HEYDARCHI, and MASOUMAH TAVASOTI KHEIRI, “CONSTRUCTION OF RECOMBINANT BACMID DNA ENCODING INFLUENZA VIRUS A (H1N1) HEMAGGLUTININ GENE,” PATHOBIOLOGY RESEARCH (MODARES JOURNAL OF MEDICAL SCIENCES), vol. 14, no. 4, pp. 38–47, 2012, [Online]. Available: https://sid.ir/paper/80953/en

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