Bahareh Rostami, Shiva Irani, Azam Bolhassani* and Reza Ahangari Cohan Pages 280 - 287 ( 8 )
Background: HIV-1 Nef protein is a possible attractive target in the development of therapeutic HIV vaccines including protein-based vaccines. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell-penetrating peptides (CPPs) as a carrier.
Methods: In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-293T mammalian cell line using a novel cell-penetrating peptide, M918, in a non-covalent fashion. The size, zeta potential and morphology of the peptide/protein complexes were studied by scanning electron microscopy (SEM) and Zeta sizer. The efficiency of Nef and Hsp20-Nef transfection using M918 was evaluated by western blotting in HEK-293T cell line.
Results: The SEM data confirmed the formation of discrete nanoparticles with a diameter of approximately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively. The dominant band of ~ 27 kDa and ~ 47 kDa was detected in the transfected cells with the Nef/ M918 and Hsp20-Nef/ M918 nanoparticles at a molar ratio of 1:20 using anti-HIV-1 Nef monoclonal antibody. These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20- Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells.
Conclusion: These data suggest that M918 CPP can be used to enter HIV-1 Nef and Hsp20-Nef proteins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.
HIV, Cell-penetrating peptide, Heat shock protein 20, Nef, M918, Transfection.
Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Pilot Nano-Biotechnology Department, Pasteur Institute of Iran, Tehran