中文摘要：研究采用TurboFect转染试剂分析了HEK-293T细胞中慢病毒载体（pCDH-CMV-MCS-EF1-cGFPT2A-puro）和真核表达载体（pEGFP-N1）表达HIV-1 Nef蛋白的效率。结果表明：这两种载体均能在靶向细胞中表达Nef蛋白，其中pEGFP-N1表达效率更高。该研究结果表明，Nef DNA和细胞穿透肽可作为Nef蛋白传递系统用于HIV疫苗制备。
外文摘要：The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFPT2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of similar to 50 kDa in SDSPAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.
外文关键词：HIV-1； Nef； Cell penetrating peptide； Transfection； In vitro gene expression； Eukaryotic and prokaryotic expression systems
作者：Jafarzade, BS; Bolhassani, A; Sadat, SM; Yaghobi, R
作者单位：伊朗巴斯德研究所
期刊名称：INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS
期刊影响因子：0.905
出版年份：2017
出版刊次：3
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