Milani A, Bolhassani A, Heshmati M
Bratisl Lek Listy. 2017;118(6):334-338. doi: 10.4149/BLL_2017_064.
Different adjuvants and delivery systems have been used to enhance the potency of DNA vaccines against viral diseases. Among them, heat shock proteins (HSPs) are stress proteins that have multiple roles such as chaperon activity and anti-apoptotic and adjuvant properties. The goal of this study was to compare the expression of HIV-1 Nef, Hsp27 and Hsp27-Nef genes transfected in HEK-293T mammalian cells by TurboFect and Lipofectamine as a cationic polymer and lipid, respectively.
At first, the pEGFP eukaryotic vectors encoding HIV-1 Nef, Hsp27 and Hsp27-Nef genes were generated and transfected in HEK-293T using TurboFect and Lipofectamine delivery systems. Then, the expression of proteins was evaluated and compared using fluorescent microscopy, flow cytometry and western blotting 48 hr after transfection.
The accuracy of the DNA constructs was confirmed on agarose gel electrophoresis to be ~ 720 bp, ~ 648 bp, and ~ 1368 bp bands for Hsp27, Nef, and Hsp-Nef, respectively. The expression analysis in the transfected cells showed that the delivery of genes using TurboFect was significantly higher than that using Lipofectamine. Furthermore, transfection of Hsp27 gene was more effective than that of Nef gene using both delivery systems. Hsp27 linked to Nef could also increase its delivery and expression in HEK-293T cells.
Generally, Hsp27 can be used as a suitable carrier in DNA vaccine design against HIV-1 infections (Fig. 5, Ref. 28).
不同的佐剂和递送系统已被用于增强针对病毒性疾病的DNA疫苗的效力。其中，热休克蛋白（HSPs）是应激蛋白，具有多种作用，如伴侣活性、抗凋亡和佐剂特性。本研究的目的是比较分别作为阳离子聚合物和脂质的TurboFect和Lipofectamine转染到HEK-293T哺乳动物细胞中的HIV-1 Nef、Hsp27和Hsp27-Nef基因的表达。
首先，构建编码HIV-1 Nef、Hsp27和Hsp27-Nef基因的pEGFP真核载体，并使用TurboFect和Lipofectamine递送系统转染到HEK-293T细胞中。然后，在转染48小时后，使用荧光显微镜、流式细胞术和蛋白质印迹法评估并比较蛋白质的表达。
在琼脂糖凝胶电泳上确认DNA构建体的准确性，Hsp27、Nef和Hsp-Nef的条带分别为720 bp、648 bp和~1368 bp。转染细胞中的表达分析表明，使用TurboFect递送基因的效率显著高于使用Lipofectamine。此外，在两种递送系统中，转染Hsp27基因比转染Nef基因更有效。与Nef连接的Hsp27也可以增加其在HEK-293T细胞中的递送和表达。
一般来说，Hsp27可作为抗HIV-1感染DNA疫苗设计中的合适载体（图5，参考文献28）。
