Volume 9, Issue 2 (6-2021)                   JoMMID 2021, 9(2): 62-70 | Back to browse issues page

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Akbari E, Ajdari S, Mirabzadeh Ardakani E, Agi E, Khalaj V, Bolhassani A. Expression of a Novel HIV-1 Gag-Pol-Env-Nef-Rev Multi-Epitope Construct in Escherichia coli. JoMMID 2021; 9 (2) :62-70
URL: http://jommid.pasteur.ac.ir/article-1-365-en.html
Expression of a Novel HIV-1 Gag-Pol-Env-Nef-Rev Multi-Epitope Construct in Escherichia coli
Elahe Akbari , Soheila Ajdari , Esmat Mirabzadeh Ardakani , Elnaz Agi , Vahid Khalaj , Azam Bolhassani
1Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran; 2Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (1613 Views)
Introduction: Recombinant subunit vaccines have been explored against various human pathogens, however, developing an effective therapeutic toward human immunodeficiency virus (HIV) infection has been challenging. So far, several recombinant HIV-1 antigens have been produced and examined for activation of desired immune responses. This study aimed to express an HIV-1 multiepitope protein as an antigen candidate to develop a vaccine.  Methods: In this study, the codon-optimized encoding sequence of the designed multi-epitope construct (Gag-Pol-Env-Nef-Rev) was synthesized and subcloned into the pET-24a (+) expression vector. Then, expression of the target antigen was evaluated in E. coli BL21 (DE3) and Rosetta strains under different conditions (temperature, optical density/ OD600, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, and time). Finally, the expression of the Gag-Pol-Env-Nef-Rev multi-epitope protein was confirmed using SDS-PAGE and western blot analysis.  Results: The highly conserved and immunodominant T-cell epitopes of HIV-1 Gag, Pol, Env, Nef, and Rev proteins were used to prepare a novel Gag-Pol-Env-Nef-Rev multi-epitope construct. The gag-pol-env-nef-rev gene was successfully sub-cloned in pET-24a (+) vector and subsequently expressed in BL21 (DE3) E. coli strain under optimized conditions (1 mM IPTG, 16 h post-induction, OD 600= 0.6, and 37ºC). A clear band of ~ 35 kDa was detected by western blotting using an anti-His antibody, indicating the successful expression of our target multi-epitope protein. Conclusion: Expression of the recombinant HIV-1 multi-epitope protein was optimized in a bacterial system. The expressed protein will be purified to use as a multi-epitope protein vaccine candidate in the future.
Keywords: Human immunodeficiency virus, Molecular cloning, Protein expression
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Type of Study: Original article | Subject: Other
Received: 2021/05/25 | Accepted: 2021/06/20 | Published: 2021/08/29
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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.