Expression of a Novel HIV-۱ Gag-Pol-Env-Nef-Rev Multi-Epitope Construct in Escherichia coli

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-۱ antigens have been produced and examined for activation of desired immune responses. This study aimed to express an HIV-۱ 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-۲۴a (+) expression vector. Then, expression of the target antigen was evaluated in E. coli BL۲۱ (DE۳) and Rosetta strains under different conditions (temperature, optical density/ OD۶۰۰, isopropyl β-D-۱-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-۱ 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-۲۴a (+) vector and subsequently expressed in BL۲۱ (DE۳) E. coli strain under optimized conditions (۱ mM IPTG, ۱۶ h post-induction, OD ۶۰۰= ۰.۶, and ۳۷ºC). A clear band of ~ ۳۵ 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-۱ 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.

کلیدواژه ها:

Human immunodeficiency virus ، Molecular cloning ، Protein expression