Sahar Sabaghzadeh, Seyed Mehdi Sadat*, Fatemeh Rohollah and Azam Bolhassani Pages 1 - 8 ( 8 )
Background: There is no effective and safe preventive/therapeutics vaccine against HIV-1 worldwide. Different viral proteins such as Nef, and two regions of Env including; variable loop of gp120 (V3) and membrane proximal external region of gp41 (MPER) are particularly important for vaccine development in different strategies and they are also the primary targets of cellular and humoral immune responses. On the other side, LDP12 is a new cell-penetrating peptide (CPP) which is capable of therapeutic application and cargoes delivery across the cellular membrane.
Objective: In current study, we designed and produced Nef-MPER-V3 fusion protein harboring LDP12 that has the capability of being used in future vaccine studies.
Methods: The CPP-protein was expressed in E.coli Rosseta (DE3) strain and purified through Ni-NTA column. Characterization of cellular delivery and toxicity of the recombinant protein were evaluated by western blotting and MTT assay.
Results: Our results showed that the CPP-protein was successfully expressed and purified with high yield of 5 mg/L. Furthermore, non-cytotoxic effect was observed and specific band (~ 37 KDa) in western blotting indicated the capability of LDP12 to improve the rate of penetration into HEK-293T cells in comparison with a control sample.
Conclusion: Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 NefMPER-V3 protein into eukaryotic cell lines without any toxicity and represented a valuable potential vaccine candidate and this guarantees the further evaluation towards the assessment of its immunogenicity in mice, which are currently under process.
Cell penetrating peptide, HIV-1, recombinant protein, Nef-MPER-V3, LDP12, vaccine.
Department of Molecular and Cellular Biology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Department of Hepatitis, AIDS and Blood borne diseases, Pasteur Institute of Iran, Tehran, Department of Molecular and Cellular Biology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Department of Hepatitis, AIDS and Blood borne diseases, Pasteur Institute of Iran, Tehran