Department of Molecular and Cellular Biology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Department of Hepatitis, AIDS and Bloodborne Diseases, Pasteur Institute of Iran, Tehran, Iran.
Protein Pept Lett. 2020;27(11):1151-1158. doi: 10.2174/0929866527666200504121400.
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.
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.
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.
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.
Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 Nef-MPER-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 is currently under process.
目前全球尚无针对 HIV-1 的有效且安全的预防性/治疗性疫苗。不同的病毒蛋白，如 Nef，以及 Env 的两个区域，包括 gp120 的可变环（V3）和 gp41 的膜近端外部区域（MPER），在不同的策略中对于疫苗开发非常重要，它们也是细胞和体液免疫反应的主要靶标。另一方面，LDP12 是一种新的细胞穿透肽（CPP），具有治疗应用和跨细胞膜输送货物的能力。
在本研究中，我们设计并生产了携带 LDP12 的 Nef-MPER-V3 融合蛋白，该蛋白有能力用于未来的疫苗研究。
CPP-蛋白在 E. coli Rosetta (DE3) 菌株中表达，并通过 Ni-NTA 柱进行纯化。通过 Western blot 和 MTT 测定评估了细胞递药和重组蛋白毒性。
我们的结果表明，CPP-蛋白成功表达并以 5mg/L 的高产量纯化。此外，Western blot 显示无细胞毒性作用，并且观察到特异性条带（~37 kDa），表明 LDP12 能够提高与对照样品相比，进入 HEK-293T 细胞的穿透率。
总之，数据表明 LDP12 CPP 可用于将 HIV-1 Nef-MPER-V3 蛋白内化到真核细胞系中，而不会产生任何毒性，并且代表了有价值的潜在疫苗候选物，这保证了进一步评估其在小鼠中的免疫原性，目前正在进行中。
