Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes
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 N...
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| Format: | Article |
| Language: | English |
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Golestan University of Medical Sciences
2021-03-01
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| Series: | Medical Laboratory Journal |
| Subjects: | |
| Online Access: | http://mlj.goums.ac.ir/article-1-1356-en.html |
| _version_ | 1818997457512562688 |
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| author | Arash Nikyar Azam Bolhassani Fatemeh Rouhollah Masoumeh Heshmati |
| author_facet | Arash Nikyar Azam Bolhassani Fatemeh Rouhollah Masoumeh Heshmati |
| author_sort | Arash Nikyar |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T21:45:56Z |
| format | Article |
| id | doaj.art-a382d3bd7b484f31bbb8791e52cb480c |
| institution | Directory Open Access Journal |
| issn | 2538-4449 |
| language | English |
| last_indexed | 2024-12-20T21:45:56Z |
| publishDate | 2021-03-01 |
| publisher | Golestan University of Medical Sciences |
| record_format | Article |
| series | Medical Laboratory Journal |
| spelling | doaj.art-a382d3bd7b484f31bbb8791e52cb480c2022-12-21T19:25:41ZengGolestan University of Medical SciencesMedical Laboratory Journal2538-44492021-03-011521117Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory GenesArash Nikyar0Azam Bolhassani1Fatemeh Rouhollah2Masoumeh Heshmati3 Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran 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.http://mlj.goums.ac.ir/article-1-1356-en.htmlhiv-1nefvprcloning |
| spellingShingle | Arash Nikyar Azam Bolhassani Fatemeh Rouhollah Masoumeh Heshmati Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes Medical Laboratory Journal hiv-1 nef vpr cloning |
| title | Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes |
| title_full | Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes |
| title_fullStr | Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes |
| title_full_unstemmed | Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes |
| title_short | Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes |
| title_sort | construction of a prokaryotic expression vector harboring two hiv 1 accessory genes |
| topic | hiv-1 nef vpr cloning |
| url | http://mlj.goums.ac.ir/article-1-1356-en.html |
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