Behzadi Mohammad Amin, Alborzi Abdolvahab, Pouladfar Gholamreza, Dianatpour Mehdi, Ziyaeyan Mazyar
Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, IR Iran.
Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran.
Jundishapur J Microbiol. 2015 Nov 26;8(11):e27355. doi: 10.5812/jjm.27355. eCollection 2015 Nov.
Although the development of novel therapeutic regimens to combat hepatitis C virus (HCV) infection have been speeded up with successful results, no efficient vaccines exist yet.
This study aimed to construct a eukaryotic expression vector encoding nonstructural proteins, NS3/NS4A, of HCV genotype 3a, and evaluate its expression on Huh7 cell surface.
The NS3/NS4A sequence was isolated from a patient with HCV-3a chronic infection, cloned into intermediate vector pTZ57R/T, and then used for engineering a mammalian expression vector, pDisplay, to direct the respective protein to the secretory pathway and anchor it to the plasma membrane. The expression of the protein in Huh7 cell, which was transiently transfected with the vector using Lipofectamine, was determined by immunocytochemical staining assay with fluorescein isothiocyanate (FITC)-conjugated antibodies to the HA/myc tags located besides the fusion fragment.
The results showed that the fragment was successfully amplified and cloned into a eukaryotic expression vector. Sequencing and enzyme digestion analysis confirmed the cloned gene completion and its correct position in the pDisply-NS3/NS4A plasmid. Immunocytochemical staining revealed that the target protein was expressed as a membrane-anchored protein in the Huh7 cells.
This study can serve as a fundamental experiment for the construction of a NS3/NS4A eukaryotic expression vector and its expression in mammalian cells. Further research is underway to evaluate the fragment immunogenicity in lab animal models.
尽管对抗丙型肝炎病毒（HCV）感染的新型治疗方案的研发已加速并取得了成功，但目前尚无有效的疫苗。
本研究旨在构建编码HCV 3a基因型非结构蛋白NS3/NS4A的真核表达载体，并评估其在Huh7细胞表面的表达。
从一名HCV-3a慢性感染患者中分离出NS³/NS4A序列，克隆到中间载体pTZ57R/T中，然后用于构建哺乳动物表达载体pDisplay，以将相应蛋白导向分泌途径并将其锚定到质膜上。使用Lipofectamine将该载体瞬时转染到Huh7细胞中，通过用针对融合片段旁HA/myc标签的异硫氰酸荧光素（FITC）偶联抗体进行免疫细胞化学染色测定来确定该蛋白在Huh7细胞中的表达。
结果表明该片段已成功扩增并克隆到真核表达载体中。测序和酶切分析证实了克隆基因的完整性及其在pDisply-NS3/NS4A质粒中的正确位置。免疫细胞化学染色显示目标蛋白在Huh7细胞中作为膜锚定蛋白表达。
本研究可为构建NS3/NS4A真核表达载体及其在哺乳动物细胞中的表达提供基础实验。正在进行进一步研究以评估该片段在实验动物模型中的免疫原性。
