作为传染性法氏囊病病毒(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大肠杆菌中提高高可溶性VP2-LS3(副猪嗜血杆菌lumazine合酶3,LS3)蛋白的表达。设计并合成了IBDVVP2-LS3基因。融合标签,GST,Nusa,MBP,ppi,γ-晶体蛋白,ArsC,和Grifin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大肠杆菌BL21(DE3)感受态细胞中。IPTG诱导后,通过SDS-PAGE和Western印迹分析来分析各种VP2-LS3蛋白的溶解度和表达水平。选择显著促进VP2-LS3蛋白的可溶性表达的融合标签。重组蛋白使用Ni-NTA亲和层析纯化,然后用TEV蛋白酶切割,用透射电子显微镜检测。凝胶电泳和测序分析表明,成功构建了7个重组载体。GST,Nusa,MBP,ppi,γ-晶体蛋白,ArsC,和Grifin增强了VP2蛋白的表达和溶解度;然而,MBP对于高纯度生产VP2-LS3更为有效。Western印迹分析证实在大肠杆菌中成功产生VP2-LS3融合蛋白。透射电镜结果显示,VP2-LS3形成了形状均匀、尺寸相对均匀的纳米颗粒。本研究建立了生成VP2-LS3重组蛋白的方法,为IBDV亚单位疫苗的开发和后续研究奠定基础。
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 E. coli. 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 E. coli 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 E. coli. 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.