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mljgoums 2021, 15(2): 11-17 |
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Nikyar A, Bolhassani A, Rouhollah F, Heshmati M. Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes. mljgoums 2021; 15 (2) :11-17
URL: http://mlj.goums.ac.ir/article-1-1356-en.html
URL: http://mlj.goums.ac.ir/article-1-1356-en.html
1- Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2- Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran ,A_bolhasani@pasteur.ac.ir
2- Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran ,
Abstract: (6346 Views)
Background and objectives: HIV-1 Nef and Vpr antigens have been described as suitable candidates for therapeutic HIV vaccine development. The aim of this study was to generate Nef-Vpr fusion gene construct and to clone the construct into pET-23a (+), a prokaryotic expression vector.
Methods: HIV-1 Nef and Vpr genes were PCR-amplified from the pNL4-3 plasmid using specific primers and Pfu DNA polymerase. Results of PCR amplification were visualized by electrophoresis on 0.8% agarose gel. At first, the amplified Nef fragment was cloned into NheI and BamHI restriction sites of pET-23a expression vector. Next, cloning of Vpr gene was performed into BamHI and HindIII restriction sites of the pET-23a-Nef vector. Finally, purity of the recombinant pET-23-Nef-Vpr construct was determined by NanoDrop spectrophotometry.
Results: PCR amplification of Nef and Vpr genes was confirmed by detection of ~ 620 bp and ~ 291 bp bands, respectively. Cloning of the Nef-Vpr construct into the vector was confirmed by detection of a ~ 911 bp fragment following enzymatic digestion with NheI and HindIII and sequencing.
Conclusion: The successful construction of recombinant fusion plasmid encoding a chimeric Nef-Vpr gene was performed in a prokaryotic expression vector for development of HIV-1 recombinant protein vaccine in near future.
Methods: HIV-1 Nef and Vpr genes were PCR-amplified from the pNL4-3 plasmid using specific primers and Pfu DNA polymerase. Results of PCR amplification were visualized by electrophoresis on 0.8% agarose gel. At first, the amplified Nef fragment was cloned into NheI and BamHI restriction sites of pET-23a expression vector. Next, cloning of Vpr gene was performed into BamHI and HindIII restriction sites of the pET-23a-Nef vector. Finally, purity of the recombinant pET-23-Nef-Vpr construct was determined by NanoDrop spectrophotometry.
Results: PCR amplification of Nef and Vpr genes was confirmed by detection of ~ 620 bp and ~ 291 bp bands, respectively. Cloning of the Nef-Vpr construct into the vector was confirmed by detection of a ~ 911 bp fragment following enzymatic digestion with NheI and HindIII and sequencing.
Conclusion: The successful construction of recombinant fusion plasmid encoding a chimeric Nef-Vpr gene was performed in a prokaryotic expression vector for development of HIV-1 recombinant protein vaccine in near future.
Research Article: Original Paper |
Subject:
Molecular Medicine
Received: 2021/01/16 | Accepted: 2021/02/24 | Published: 2021/02/28 | ePublished: 2021/02/28
Received: 2021/01/16 | Accepted: 2021/02/24 | Published: 2021/02/28 | ePublished: 2021/02/28
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