Virology Department, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Virology Department, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Microb Pathog. 2018 May;118:87-90. doi: 10.1016/j.micpath.2018.03.016. Epub 2018 Mar 9.
Flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system.
The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR.
The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine.
The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.
鞭毛蛋白是细菌鞭毛的主要结构蛋白单体。鞭毛蛋白通过与受体结合并激活抗原呈递细胞，刺激先天和适应性免疫反应。鞭毛蛋白被用作有效的全身或粘膜佐剂来刺激免疫系统。最近，鞭毛蛋白在一些传染病和癌症中的治疗和保护作用已经被研究。在这项研究中，我们从鼠伤寒沙门氏菌和大肠杆菌中克隆了 fliC 基因到 pET-28a 载体中，并在原核系统中研究了它们的表达。
使用特定的寡核苷酸引物对 PCR 扩增鼠伤寒沙门氏菌和大肠杆菌的 fliC 基因。thse 被克隆到 pET-28a 载体中，重组 pET-28a-fliC 质粒成功转化到大肠杆菌菌株 BL-21(DE3)中。在原核细胞中评估鞭毛蛋白的表达。最后，使用实时 PCR 确认 TNF-αmRNA 的转录。
通过 SDS-PAGE 和 Western blot 方法证实了原核细胞中蛋白质的表达。此外，通过评估其诱导促炎细胞因子 m-RNA 表达增加的能力来评价鞭毛蛋白的功能特征。
鞭毛蛋白在原核系统中表达。这些蛋白质可用于连接靶抗原，作为未来 DNA 疫苗研究的有效佐剂。本研究中纯化的重组蛋白也可用于治疗和预防目的。
