An efficient method for detection of foot and mouth disease (FMD) and, particularly, differentiation of vaccinated from infected animals is the use of nonstructural (NS) proteins as antigens in Enzyme-Linked Immunosorbent Assay (ELISA) Kits. In this study, only epitopic regions of 3AB and 3D NS proteins were used for recombinant protein production, as a cost-effective method instead of peptide synthesis, for application in in-house ELISA diagnostic kits. Specific primers were designed according to the antigenic regions of 3AB (C-terminus of 3A and the whole 3B) and 3D (N-terminus) proteins, and the polymerase chain reaction (PCR) amplification was performed. Purified amplicons were cloned into pET21a (+) vectors and then transformed into Escherichia coli (BL21). Thereafter, bacteria were induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for expression of antigenic proteins. Antigenic 3AB protein was expressed in soluble form, but 3D protein was extracted from the bacterial lysate. Protein expression was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analyses. An indirect ELISA was developed for each protein, and the diagnostic sensitivity and specificity were determined. The 3AB-ELISA showed higher sensitivity and specificity than 3D-ELISA (95.24% and 100%, compared with 90.48% and 88.71%, respectively). The epitopic 3AB-ELISA developed here can be used for detection and differentiation of FMD infected from vaccinated animals, but the epitopic 3D-ELISA showed lower efficiency in screening for FMD status.

中文翻译：

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3AB 和 3D 非结构蛋白的表位位点是否足以检测口蹄疫？

检测口蹄疫 (FMD) 的一种有效方法，特别是区分已接种疫苗的动物和感染动物的方法是在酶联免疫吸附试验 (ELISA) 试剂盒中使用非结构 (NS) 蛋白作为抗原。在本研究中，仅将 3AB 和 3D NS 蛋白的表位区域用于重组蛋白生产，作为一种经济有效的方法，而不是肽合成，用于内部 ELISA 诊断试剂盒。根据3AB（3A的C端和整个3B）和3D（N端）蛋白的抗原区设计特异性引物，进行聚合酶链式反应（PCR）扩增。将纯化的扩增子克隆到 pET21a (+) 载体中，然后转化到大肠杆菌中(BL21)。此后，用 1 mM 异丙基 β-d-1-硫代吡喃半乳糖苷 (IPTG) 诱导细菌表达抗原蛋白。抗原性 3AB 蛋白以可溶形式表达，但 3D 蛋白是从细菌裂解物中提取的。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹分析确认蛋白质表达。为每种蛋白质开发了间接ELISA，并确定了诊断敏感性和特异性。3AB-ELISA 显示出比 3D-ELISA 更高的灵敏度和特异性（分别为 95.24% 和 100%，而分别为 90.48% 和 88.71%）。此处开发的表位 3AB-ELISA 可用于检测和区分接种动物感染的口蹄疫，但表位 3D-ELISA 在筛查口蹄疫状态方面的效率较低。