College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China.
Cell Cycle. 2022 Jul;21(14):1532-1542. doi: 10.1080/15384101.2022.2056305. Epub 2022 Mar 28.
As part of the development of an infectious bursal disease virus (IBDV) subunit vaccine, this study was designed to improve the expression of highly soluble VP2-LS3 (Haemophilus parasuis lumazine synthase 3, LS3) protein by using different tagged vectors in . IBDV VP2-LS3 gene was designed and synthesized. Fusion tags, GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin were joined to the N-terminus of VP2-LS3 protein. Seven expression plasmids were constructed, and each plasmid was transformed into BL21 (DE3) competent cells. After induction by IPTG, the solubility and expression levels of the various VP2-LS3 proteins were analyzed by SDS-PAGE and Western Blot analysis. The fusion tag that significantly promoted soluble expression of the VP2-LS3 protein was selected. Recombinant proteins were purified using Ni-NTA affinity chromatography, then cleaved by using TEV protease and detected by using transmission electron microscopy. Gel electrophoresis and sequencing analysis showed that all seven recombinant vectors were successfully constructed. GST, NusA, MBP, Ppi, γ-crystallin, ArsC, and Grifin enhanced the expression and solubility of VP2 protein; however, MBP was more effective for the high-purity production of VP2-LS3. Western Blot analysis confirmed successful generation of VP2-LS3 fusion protein in . The result of transmission electron microscopy showed that VP2-LS3 formed nano-sized particles with homogeneous shape and relatively uniform size. This study established a method to generate VP2-LS3 recombinant protein, which may lay a foundation for the development and subsequent study of IBDV subunit vaccines.
作为传染性腔上囊病病毒 (IBDV) 亚单位疫苗开发的一部分，本研究旨在通过使用不同的标记载体在 中提高高度可溶性 VP2-LS3（副猪嗜血杆菌尿囊素合酶 3，LS3）蛋白的表达。设计并合成了 IBDV VP2-LS3 基因。融合标签 GST、NusA、MBP、Ppi、γ-晶体蛋白、ArsC 和 Griffin 被连接到 VP2-LS3 蛋白的 N 端。构建了七个表达质粒，并将每个质粒转化到 BL21 (DE3) 感受态细胞中。用 IPTG 诱导后，通过 SDS-PAGE 和 Western Blot 分析来分析各种 VP2-LS3 蛋白的可溶性表达和表达水平。选择了显著促进 VP2-LS3 蛋白可溶性表达的融合标签。使用 Ni-NTA 亲和层析纯化重组蛋白，然后用 TEV 蛋白酶切割，并使用透射电子显微镜检测。凝胶电泳和测序分析表明，所有七个重组载体均成功构建。GST、NusA、MBP、Ppi、γ-晶体蛋白、ArsC 和 Griffin 增强了 VP2 蛋白的表达和可溶性；然而，MBP 更有利于 VP2-LS3 的高纯度生产。Western Blot 分析证实了 在 中成功产生了 VP2-LS3 融合蛋白。透射电子显微镜的结果表明，VP2-LS3 形成了具有均匀形状和相对均匀尺寸的纳米级颗粒。本研究建立了生成 VP2-LS3 重组蛋白的方法，这可能为 IBDV 亚单位疫苗的开发和后续研究奠定基础。
