Expression of a Novel HIV-1 Gag-Pol-Env-Nef-Rev Multi-Epitope Construct in Escherichia coli

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. 

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. 

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.

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.