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利用新型细胞穿透肽设计和体外递呈 HIV-1 多表位 DNA 和肽构建体。

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides.

机构信息

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.

出版信息

Biotechnol Lett. 2019 Nov;41(11):1283-1298. doi: 10.1007/s10529-019-02734-x. Epub 2019 Sep 17.

Abstract

OBJECTIVES

Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system.

RESULTS

Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef, Nef, Vpr, Vpr, Gp160, Gp160 and P24 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32 kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293 T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200 nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293 T cells.

CONCLUSION

The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

摘要

目的

由于 HIV-1 病毒的多样性、人类 HLA 的多态性以及缺乏合适的传递系统,开发能够刺激体液和细胞免疫反应的有效 HIV 疫苗仍然具有挑战性。

结果

我们使用生物信息学工具设计了一种编码多个表位的 DNA 构建体。这些表位在流行的 HIV-1 亚型内高度保守,并与伊朗和世界上流行的 I 类和 II 类 HLA 相互作用。设计的 DNA 构建体包括 Nef、Nef、Vpr、Vpr、Gp160、Gp160 和 P24 表位(即 nef-vpr-gp160-p24 DNA),这些表位被克隆到 pET-24a(+)和 pEGFP-N1 载体中。重组多表位肽(rNef-Vpr-Gp160-P24;~32 kDa)在大肠杆菌表达系统中成功生成。pEGFP-nef-vpr-gp160-p24 和 rNef-Vpr-Gp160-P24 多表位肽通过细胞穿透肽(CPP)递送至 HEK-293 T 细胞。MPG 和 HR9 CPP 以及新型 LDP-NLS 和 CyLoP-1 CPP 分别用于将 DNA 和肽递送至细胞。SEM 结果证实了稳定的 MPG/pEGFP-N1-nef-vpr-gp160-p24、HR9/pEGFP-N1-nef-vpr-gp160-p24、LDP-NLS/rNef-Vpr-Gp160-P24 和 CyLoP-1/rNef-Vpr-Gp160-P24 纳米颗粒的形成,这些纳米颗粒通过非共价键形成,直径<200nm。MTT 测定结果表明,这些纳米颗粒在体外无明显毒性。荧光显微镜、流式细胞术和 Western blot 数据表明,这些 CPP 可以显著将 DNA 和肽构建体递送至 HEK-293 T 细胞。

结论

这些 CPP 的使用可以被认为是一种 HIV 疫苗开发方法,用于将 DNA 和肽构建体递送至哺乳动物细胞的体外和体内。

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