Medical Laboratory Journal
Medical Laboratory Journal
mljgoums
Medical Sciences
http://mlj.goums.ac.ir
1
admin
2538-4449
10.52547/mlj
en
jalali
1399
12
1
gregorian
2021
3
1
15
2
online
1
fulltext
en
Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes
پزشکی مولکولی
Molecular Medicine
تحقيقي
Original Paper
<strong>Background and objectives:</strong> 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 <em>Nef-Vpr</em> fusion gene construct and to clone the construct into pET-23a (+), a prokaryotic expression vector.<br>
<strong>Methods:</strong> HIV-1 <em>Nef </em>and <em>Vpr</em> genes were PCR-amplified from the <em>pNL4</em>-<em>3</em> 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 <em>Nhe</em>I and <em>BamHI</em> restriction sites of pET-23a expression vector. Next, cloning of <em>Vpr</em> gene was performed into <em>Bam</em>HI and <em>Hind</em>III restriction sites of the pET-23a-Nef vector. Finally, purity of the recombinant pET-23-<em>Nef-Vpr</em> construct was determined by NanoDrop spectrophotometry.<br>
<strong>Results:</strong> PCR amplification of <em>Nef</em> and <em>Vpr</em> genes was confirmed by detection of ~ 620 bp and ~ 291 bp bands, respectively. Cloning of the <em>Nef-Vpr</em> construct into the vector was confirmed by detection of a ~ 911 bp fragment following enzymatic digestion with <em>Nhe</em>I and <em>Hin</em>dIII and sequencing.<br>
<strong>Conclusion: </strong>The successful construction of recombinant fusion plasmid encoding a chimeric <em>Nef-Vpr</em> gene was performed in a prokaryotic expression vector for development of HIV-1 recombinant protein vaccine in near future.
HIV-1,Nef,Vpr,Cloning,
11
17
http://mlj.goums.ac.ir/browse.php?a_code=A-10-640-1&slc_lang=en&sid=1
Arash
Nikyar
arashnikyar@yahoo.com
100319475328460021159
100319475328460021159
No
Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
Azam
Bolhassani
A_bolhasani@pasteur.ac.ir
100319475328460021160
100319475328460021160
Yes
Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran
Fatemeh
Rouhollah
100319475328460021161
100319475328460021161
No
Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
Masoumeh
Heshmati
100319475328460021162
100319475328460021162
No
Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran