Volume 19, Issue 4 (7-2016)                   J Arak Uni Med Sci 2016, 19(4): 60-68 | Back to browse issues page

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Kadkhodayan S, Irani S, Sadat S M, Fotouhi F, Bolhassani A. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System. J Arak Uni Med Sci 2016; 19 (4) :60-68
URL: http://jams.arakmu.ac.ir/article-1-4231-en.html
1- Department of Biology, Sciences and Research Branch, Islamic Azad University, Tehran, Iran.
2- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
3- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
4- Influenza Research Lab., Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
5- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. , A_bolhasani@pasteur.ac.ir
Abstract:   (6710 Views)

Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method.

Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3) strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method.

Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it.

Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

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Type of Study: Original Atricle | Subject: General
Received: 2016/02/6 | Accepted: 2016/04/13

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