Journal of Medical Microbiology and Infectious Diseases
Journal of Medical Microbiology and Infectious Diseases
JoMMID
Medical Sciences
http://jommid.pasteur.ac.ir
1
admin
2345-5349
2345-5330
8
10.61882/JoMMID
14
8888
13
en
jalali
1400
3
1
gregorian
2021
6
1
9
2
online
1
fulltext
en
Expression of a Novel HIV-1 Gag-Pol-Env-Nef-Rev Multi-Epitope Construct in Escherichia coli
Other
Other
Original article
Original article
<strong>Introduction: </strong>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. <strong>Methods: </strong>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 <em>E. coli</em> BL21 (DE3) and Rosetta strains under different conditions (temperature, optical density/ OD<sub>600</sub>, 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. <strong>Results: </strong>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 <em>gag-pol-env-nef-rev</em> gene was successfully sub-cloned in pET-24a (+) vector and subsequently expressed in BL21 (DE3) <em>E. coli </em>strain under optimized conditions (1 mM IPTG, 16 h post-induction, OD <sub>600</sub>= 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. <strong>Conclusion: </strong>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.
Human immunodeficiency virus, Molecular cloning, Protein expression
62
70
http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-123-7&slc_lang=en&sid=1
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