Volume 15, Issue 2 (Mar-Apr 2021)                   mljgoums 2021, 15(2): 11-17 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

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
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
Abstract:   (1190 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.
Keywords: HIV-1 [MeSH], Nef [MeSH], Vpr , Cloning [MeSH],
Full-Text [PDF 895 kb]   (207 Downloads) |   |   Full-Text (HTML)  (256 Views)  
Research Article: Original Paper | Subject: Molecular Medicine
Received: 2021/01/16 | Accepted: 2021/02/24 | Published: 2021/02/28 | ePublished: 2021/02/28

1. German Advisory Committee Blood (Arbeitskreis Blut), Subgroup 'Assessment of Pathogens Transmissible by Blood' GACB (Arbeitskreis, Blood' S 'Assessment of PT by. Human Immunodeficiency Virus (HIV). Transfus Med Hemother. 2016; 43(3): 203-22. [DOI:10.1159/000445852] [PubMed]
2. Global HIV, Hepatitis and STIs Programme. [cited 2021 Jan 8]. Available from: https://www.who.int/teams/global-hiv-hepatitis-and-stis-programmes/data-use/hiv-data-and-statistics [View at Publisher]
3. Li G, De Clercq E. HIV Genome-Wide Protein Associations: a Review of 30 Years of Research. Microbiol Mol Biol Rev. 2016; 80(3): 679-731. [DOI:10.1128/MMBR.00065-15] [PubMed] [Google Scholar]
4. Tristem M, Purvis A, Quicke DLJ. Complex evolutionary history of primate lentiviral vpr genes. Virology. 1998 Jan 15;240(2):232-7. [DOI:10.1006/viro.1997.8929] [PubMed] [Google Scholar]
5. Guenzel CA, Hérate C, Benichou S. HIV-1 Vpr-a still "enigmatic multitasker. Front Microbiol. 2014; 5: 127. [View at Publisher] [DOI:10.3389/fmicb.2014.00127] [PubMed] [Google Scholar]
6. Lubow J, Collins KL. Vpr Is a VIP: HIV Vpr and Infected Macrophages Promote Viral Pathogenesis. Viruses. 2020; 12(8): 809. [View at Publisher] [DOI:10.3390/v12080809] [PubMed] [Google Scholar]
7. Zhao RY, Li G, Bukrinsky MI. Vpr-Host Interactions During HIV-1 Viral Life Cycle. J Neuroimmune Pharmacol. 2011; 6(2): 216-29. [DOI:10.1007/s11481-011-9261-z] [PubMed] [Google Scholar]
8. Basmaciogullari S, Pizzato M. The activity of Nef on HIV-1 infectivity. Front Microbiol. 2014; 5(MAY):232. [View at Publisher] [DOI:10.3389/fmicb.2014.00232] [PubMed] [Google Scholar]
9. Ferdin J, Goričar K, Dolžan V, Plemenitaš A, Martin JN, Peterlin BM, et al. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA. PLoS One. 2018; 13(1): e0191613. [DOI:10.1371/journal.pone.0191613] [PubMed] [Google Scholar]
10. Da-Yong-Lu, Wu H-Y, Yarla NS, Xu B, Ding J, Lu T-R. HAART in HIV / AIDS Treatments , a Current Limitation. EC Orthop. 2017; 4(Table 1): 140-6. [Google Scholar]
11. Nascimento IP, Leite LCC. Recombinant vaccines and the development of new vaccine strategies [Internet]. Vol. 45, Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica; 2012 [cited 2021 Jan 2]. p. 1102-11. Available from: /pmc/articles/PMC3854212/?report=abstract [DOI:10.1590/S0100-879X2012007500142] [PubMed] [Google Scholar]
12. Daemi A, Hosseinzadeh S, Rafati S, Zahedifard F, Rajabi Bazl M, Doustdari F, et al. HPV16 E7-CT (gp96) fusion protein: Molecular cloning, expression and purification of a recombinant 6xHis-tagged protein in E. coli. J Paramed Sci. 2012; 3(2): 2-7. [View at Publisher] [Google Scholar]
13. Pandey A, Galvani AP. The global burden of HIV and prospects for controlLancet HIV. 2019; 6(12): e809-e811. [DOI:10.1016/S2352-3018(19)30230-9] [PubMed] [Google Scholar]
14. Wen J, Hao W, Fan Y, Du J, Du B, Qian M, et al. Co-delivery of LIGHT expression plasmid enhances humoral and cellular immune responses to HIV-1 Nef in mice. Arch Virol. 2014; 159(7): 1663-9. [View at Publisher] [DOI:10.1007/s00705-014-1981-y] [PubMed] [Google Scholar]
15. Tähtinen M, Strengell M, Collings A, Pitkänen J, Kjerrström A, Hakkarainen K, et al. DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector. Vaccine. 2001; 19(15-16): 2039-47. [View at Publisher] [DOI:10.1016/S0264-410X(00)00420-5] [PubMed] [Google Scholar]
16. Jafarzade BS, Bolhassani A, Sadat SM, Yaghobi R. Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine. Int J Pept Res Ther. 2017; 23(1): 145-53. [View at Publisher] [DOI:10.1007/s10989-016-9547-3] [Google Scholar]
17. Xie XY, Wan YM, Li B, Shi JJ, Qiu C, Liu ZQ, et al. Immunogenicity of DNA vaccines encoding regulatory/accessory proteins derived from three different prevalent strains in China. Chinese J Microbiol Immunol. 2011; 31(2): 157-61. [Google Scholar]
18. Gonzalez ME. The HIV-1 vpr protein: A multifaceted target for therapeutic intervention. International Journal of Molecular Sciences. MDPI AG. 2017; 73(5): 4101-9. [DOI:10.3390/ijms18010126] [PubMed] [Google Scholar]
19. Hrimech M, Yao X-J, Bachand F, Rougeau N, Cohen ÉA. Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Functions as an Immediate-Early Protein during HIV-1 Infection. J Virol [Internet]. 1999; 73(5): 4101-9. [DOI:10.1128/JVI.73.5.4101-4109.1999] [PubMed] [Google Scholar]
20. Miller RH, Sarver N. HIV accessory proteins as therapeutic targets. Nat Med. 1997; 3(4): 389-94. [DOI:10.1038/nm0497-389] [PubMed] [Google Scholar]
21. Kostylev M, Otwell AE, Richardson RE, Suzuki Y. Cloning should be simple: Escherichia coli DH5á-mediated assembly of multiple DNA fragments with short end homologies. PLoS One. 2015; 10(9): e0137466. [DOI:10.1371/journal.pone.0137466] [PubMed] [Google Scholar]
22. Phue JN, Sang JL, Trinh L, Shiloach J. Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α). Biotechnol Bioeng. 2008; 101(4): 831-6. [DOI:10.1002/bit.21973] [PubMed] [Google Scholar]
23. Dobrijevic D, Nematollahi LA, Hailes HC, Ward JM. pET expression vector customized for efficient seamless cloning. Biotechniques. 2020; 69(5): 384-7. [View at Publisher] [DOI:10.2144/btn-2020-0101] [PubMed] [Google Scholar]
24. Gay G, Wagner DT, Keatinge-Clay AT, Gay DC. Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid. 2014; 76: 66-71. [View at Publisher] [DOI:10.1016/j.plasmid.2014.09.005] [PubMed] [Google Scholar]
25. Davoodi S, Bolhassani A, Sadat SM, Irani S. Enhancing HIV-1 Nef Penetration into Mammalian Cells as an Antigen Candidate. J Med Microbiol Infect Dis. 2019; 7(1): 37-43. [View at Publisher] [DOI:10.29252/JoMMID.] [PubMed] [Google Scholar]
26. Davoodi S, Bolhassani A, Sadat SM, Irani S. Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides. Biotechnol Lett. 2019; 41(11): 1283-98. [View at Publisher] [DOI:10.1007/s10529-019-02734-x] [PubMed] [Google Scholar]
27. Elbahnasawy MA, Farag MMS, Mansour MT, El-Ghamery AA. Cloning, expression and nanodiscs assemble of recombinant HIV-1 gp41. Microb Pathog. 2020;138. [DOI:10.1016/j.micpath.2019.103824] [PubMed] [Google Scholar]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2007 All Rights Reserved | Medical Laboratory Journal

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.