Mechnikov Scientific Research Institute of Vaccines and Sera, 5A Maly Kazenny Lane, Moscow, 105064, Russia.
FSBIS G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Protein Expr Purif. 2021 Jul;183:105864. doi: 10.1016/j.pep.2021.105864. Epub 2021 Mar 4.
In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.
在这项研究中，我们描述了一种从酵母细胞（毕赤酵母）中表达的重组丙型肝炎病毒核心蛋白（HCcAg）获得病毒样颗粒（VLPs）的优化方法，该方法可用于构建诊断测试系统和疫苗工程。所描述的简化程序是为了在蛋白质纯化过程中使 HCcAg 分子在体外自组装成 VLPs。简而言之，将 HCcAg 蛋白从酵母细胞裂解物中用硫酸铵沉淀，并在还原条件下通过 Sephadex G-25 凝胶过滤复性。通过 Sephadex G-25 凝胶过滤去除还原剂后，自组装 VLPs。通过 SDS-PAGE 和免疫印迹分析评估蛋白质纯度和特异性。通过 HPLC 测量 VLPs 的分子量及其相对数量，并通过透射电子显微镜确认 VLPs 的产生以及对其形状和大小的估计。结果，我们以 VLPs 和单体的形式获得了重组 HCcAg 制剂（纯度约为 90%），并已用于生产针对 HCcAg 的杂交瘤分泌单克隆抗体（mAbs）。
