Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China.
J Biotechnol. 2012 Oct 31;161(3):221-7. doi: 10.1016/j.jbiotec.2012.05.004. Epub 2012 May 23.
The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.
E2 包膜糖蛋白是经典猪瘟病毒（CSFV）的主要免疫优势蛋白，可诱导感染猪产生中和抗体和保护性免疫应答。我们开发了一种串联重复的多表位重组蛋白，该蛋白包含 E2 蛋白的两个拷贝，分别跨越残基 693-704、770-780 和 826-843，由 CSFV 非结构蛋白 NS2-3 的残基 1446-1460 跨越的两个拷贝连接。该化学合成基因在大肠杆菌中作为谷胱甘肽 S-8（GST）的融合体表达，命名为 GST-BT21。在用 Glutathione Sepharose 4B 纯化后，我们使用 Western blot 来表征构建体，并使用表面等离子体共振来分析其与 CSFV 阳性血清的亲和力和特异性相互作用。纯化的 GST-BT21 蛋白与针对 CSFV 的抗血清显示出极好的免疫反应性（Tian 等人，2012 年），表面等离子体共振证实了 BT21 与感染 CSFV 的动物血清中的抗体之间的特异性亲和力，而不是 GST。表面等离子体共振是一种敏感而精确的表位评估方法，可用于表征重组蛋白的免疫原性和功能。
