Doctoral Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung, Taiwan.
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Appl Microbiol Biotechnol. 2024 Apr 19;108(1):303. doi: 10.1007/s00253-024-13135-0.
Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SS, derived from the extension protein, and the (SP) motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.
严重发热伴血小板减少综合征病毒(SFTSV)可导致人类罹患致命疾病。为了便于诊断,本研究利用基于竹嵌纹病毒(Bamboo mosaic virus,BaMV)的载体系统,在本氏烟中产生了天然形式的亚单位糖蛋白(glycoprotein,Gn),该蛋白是潜在疫苗和疗法的主要靶标。通过与来自延伸蛋白的分泌信号标签 SS 和(SP)基序融合,重组 Gn(recombinant Gn,rGn)的产量显著增加,达到约 7mg/kg 侵染叶片。最终,成功建立了基于 rGn 的 ELISA 法,用于检测自然感染猴血清样本中的 SFTSV 特异性抗体。该方法与参考方法验证的结果一致,rGn-ELISA 的特异性和灵敏度分别为 94%和 96%。总之,利用合适的融合标签可大量生产和纯化 rGn ,同时保持其抗原性。rGn-ELISA 具有良好的灵敏度和特异性,可作为评估 SFTSV 血清流行率的一种可行替代诊断方法。关键点: • 与分泌信号标签融合的 SFTSV Gn 由 BaMV 载体表达。 • 植物融合标签提高了 rGn 的表达水平并简化了其纯化过程。 • 建立并验证了 rGn-ELISA,其特异性和灵敏度均>94%。