Department of Hepatitis, AIDS and Blood Borne Diseases, Pasteur Institute of Iran, Tehran, Iran.
Department of Arboviruses and Viral Hemorrhagic Fevers (National Ref Lab), Pasteur Institute of Iran, Tehran, Iran.
Curr Drug Deliv. 2020;17(5):387-395. doi: 10.2174/1567201817666200317121728.
Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens' features before applying them.
This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1.
The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction.
Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression.
Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.
尽管尚未完全实用，但已经研究了不同的方法来开发预防性或治疗性疫苗。针对 HIV-1 的治疗性疫苗的研究旨在通过或不通过高效抗逆转录病毒疗法 (HAART) 从储库细胞中消除病毒。具有保守区域中最具免疫原性特征的融合蛋白可以促进这种在如此多变的病毒中的实现。为了获得最具免疫原性和保守性的区域，生物信息学工具广泛用于在应用之前预测抗原的特征。
本研究旨在体外评估基于先前计算机设计的 p24-Nef 融合蛋白，以获得针对 HIV-1 的潜在治疗性亚单位疫苗。
使用 AAY 柔性接头表达和评估截短形式的 p24-Nef 和全长蛋白，并通过 Western blot 进行验证。我们还使用 pcDNA3.1 转染 Lenti-X 293T 细胞。此外，应用慢病毒载体生产携带感兴趣基因的重组病毒粒子，并进行细胞转导。
两种融合蛋白（截短和全长形式）均通过 Western blot 中的抗 Nef 多克隆抗体表达和确认。生成重组病毒粒子并转导 Lenti-X 293T 细胞，通过免疫荧光显微镜和 p24 ELISA 试剂盒进行确认。转导细胞通过 SDS-PAGE 和 Western blot 进行分析，结果表明表达了批准的蛋白。
根据生物信息学工具预先评估的特征，融合蛋白 p24 和 Nef 在真核细胞系中得到了很好的表达。
