转染
分子生物学
免疫原性
流式细胞术
重组DNA
肽
免疫印迹
DNA
活力测定
HEK 293细胞
MTT法
细胞毒性T细胞
体外
化学
生物
基因
免疫系统
生物化学
免疫学
作者
Arash Nikyar,Azam Bolhassani,Fatemeh Rouhollah,Masoumeh Heshmati
出处
期刊:Current Drug Delivery
[Bentham Science]
日期:2022-12-01
卷期号:19 (10): 1083-1092
被引量:1
标识
DOI:10.2174/1567201819666220217164055
摘要
DNA-based therapeutic vaccines have been proposed as a promising strategy for the treatment of established HIV infections. However, these vaccines are often associated with certain shortcomings, such as poor immunogenicity and low transfection efficiency. In this study, we investigated the ability of LL-37 to deliver a potential immunogenic fusion construct comprising HIV-1 nef and vpr genes into a mammalian cell line.First, the pEGFP-N1 eukaryotic expression vector harboring the HIV-1 nef-vpr fusion was produced free of endotoxin on a large scale. Then, DNA/LL-37 complexes were prepared by coincubation of pEGFP-nef-vpr with LL-37 for 45 minutes at different nitrogen to phosphate (N/P) ratios. The formation of DNA/peptide complexes was investigated by gel retardation assay. Next, the stability and morphological characteristics of the nanoparticles were evaluated. The toxicity of LL-37 and the nanoparticles in HEK-293T cells were assessed by MTT assay. The transfection efficiency of the DNA/LL-37 complexes was studied by fluorescence microscopy, flow cytometry, and western blot analysis.LL-37 formed stable complexes with pEGFP-nef-vpr (diameter of 150-200 nm) while providing good protection against nucleolytic and proteolytic degradation. The peptide significantly affected cell viability even at low concentrations. However, the LL-37/DNA complexes had no significant cytotoxic effect. Treatment of cells with pEGFP-N1/LL-37 and pEGFP-nef-vpr/LL-37 resulted in transfection of 36.32% ± 1.13 and 25.55% ± 2.07 of cells, respectively.Given these findings and the important immunomodulatory and antiviral activities of LL- 37, the use of this peptide can be further exploited in the development of novel gene delivery strategies and vaccine design.
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