Taherkhani Reza, Farshadpour Fatemeh, Makvandi Manoochehr
Department of Microbiology and Parasitology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran; Persian Gulf Biomedical Research Center, Bushehr University of Medical Sciences, Bushehr, Iran.
J Med Virol. 2015 Jul;87(7):1225-34. doi: 10.1002/jmv.24171. Epub 2015 Mar 17.
The aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against Hepatitis E virus (HEV). Initially, conserved and antigenic helper T-lymphocyte (HTL) epitopes in the HEV capsid protein were predicted by in silico analysis. Subsequently, a multiepitope comprising four HTL epitopes with high-affinity binding to the HLA molecules was designed, and repeated four times as high density multiepitope construct. This construct was synthesized and cloned into pET-30a (+) vector. Then, it was transformed and expressed in Escherichia coli BL21 cells. The high density multiepitope protein was purified by Ni-NTA agarose and concentrated using Amicon filters. Finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. The results showed that the high density multiepitope construct was successfully expressed and purified. SDS-PAGE and Western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kDa. Approximately 1 mg of the purified protein was obtained from each liter of the culture media. Moreover, the purified multiepitope protein was capable of induction of proliferation responses, IFN-γ ELISPOT responses and IFN-γ and IL-12 cytokines production in a significant level in peripheral blood mononuclear cells (PBMCs) isolated from HEV-recovered individuals compared to the control group. In conclusion, the newly produced multiepitope protein can induce significant T helper type 1 responses in vitro, and can be considered as a novel strategy for the development of HEV vaccines in the future.
本研究的目的是设计一种高密度多表位蛋白，它有望成为抗戊型肝炎病毒（HEV）的多表位疫苗候选物。最初，通过计算机分析预测了HEV衣壳蛋白中保守且具有抗原性的辅助性T淋巴细胞（HTL）表位。随后，设计了一个包含四个与HLA分子具有高亲和力结合的HTL表位的多表位，并作为高密度多表位构建体重复四次。该构建体被合成并克隆到pET-30a(+)载体中。然后，将其转化并在大肠杆菌BL21细胞中表达。高密度多表位蛋白通过Ni-NTA琼脂糖纯化，并使用Amicon过滤器浓缩。最后，在体外评估了这种高密度多表位蛋白的免疫特性。结果表明，高密度多表位构建体成功表达并纯化。SDS-PAGE和蛋白质印迹分析显示存在一条约33 kDa的高密度多表位蛋白条带。每升培养基可获得约1 mg的纯化蛋白。此外，与对照组相比，纯化的多表位蛋白能够在从HEV康复个体分离的外周血单核细胞（PBMC）中显著诱导增殖反应、IFN-γ ELISPOT反应以及IFN-γ和IL-12细胞因子的产生。总之，新产生的多表位蛋白在体外可诱导显著的1型辅助性T细胞反应，可被视为未来开发HEV疫苗的一种新策略。
