Kadkhodayan Somayeh, Bolhassani Azam, Sadat Seyed Mehdi, Irani Shiva, Fotouhi Fatemeh
Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Hepatitis and AIDs Pasteur Institute of Iran, Tehran, Iran.
Curr Drug Deliv. 2017;14(4):536-542. doi: 10.2174/1567201813666161006114448.
Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane.
In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 Tat- Nef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E. coli and transfected using two amphipathic CPPs (Pep-1 and Cady-2) into mammalian cells. The size and morphology of the CPP/GST-Tat-Nef complexes were evaluated by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The transfection of HIV-1 GST-Tat-Nef protein was also analyzed using SDS-PAGE and western blotting.
Our data indicated that the recombinant GST-Tat-Nef protein generated in BL21 strain migrated as a clear band of ~ 52 kDa in SDS-PAGE. The results of SEM and Zetasizer confirmed the formation of protein/ Pep-1 or protein/ Cady-2 nanoparticles less than 200 nm in diameter. Tat CPP fused to Nef protein could deliver the recombinant Nef protein alone and notably by forming the noncovalent complexes with TurboFect, Pep-1 and Cady-2 as detected in western blotting. Moreover, intracellular uptake of Tat-Nef gene and subsequently its expression in mammalian cells was considerably higher than that for Nef gene.
This data indicated that the Tat gene sequence could also increase the transfection of Nef gene in vitro. Generally, the Tat-Nef interaction led to enhance further gene expression and also protein delivery.
细胞穿透肽（CPPs）或蛋白质转导结构域（PTDs）已成为货物递送领域的一个新方向。这些肽，如Tat、Pep-1和Cady-2，能够通过质膜将基因和生物活性蛋白递送至细胞质区室。
在本研究中，使用TurboFect转染试剂在HEK-293T细胞中评估了pEGFP-N1真核载体表达HIV-1 Tat-Nef融合蛋白的效率。此外，在大肠杆菌中产生重组GST-Tat-Nef蛋白，并使用两种两亲性CPPs（Pep-1和Cady-2）将其转染到哺乳动物细胞中。通过扫描电子显微镜（SEM）、Zetasizer和SDS-PAGE评估CPP/GST-Tat-Nef复合物的大小和形态。还使用SDS-PAGE和蛋白质印迹法分析HIV-1 GST-Tat-Nef蛋白的转染情况。
我们的数据表明，在BL21菌株中产生的重组GST-Tat-Nef蛋白在SDS-PAGE中迁移为一条清晰的~52 kDa条带。SEM和Zetasizer的结果证实形成了直径小于200 nm的蛋白质/Pep-1或蛋白质/Cady-2纳米颗粒。与Nef蛋白融合的Tat CPP能够单独递送重组Nef蛋白，并且如在蛋白质印迹法中检测到的，通过与TurboFect、Pep-1和Cady-2形成非共价复合物，递送效果显著。此外，Tat-Nef基因在哺乳动物细胞中的细胞内摄取以及随后的表达明显高于Nef基因。
该数据表明Tat基因序列在体外也可提高Nef基因的转染效率。一般来说，Tat-Nef相互作用导致进一步增强基因表达以及蛋白质递送。
