Campus Colemar Natal E Silva, Instituto de Patologia Tropical E Saúde Pública, Universidade Federal de Goiás, Goiânia, GO, 74605-450, Brazil.
Faculdade de Farmácia, Campus Colemar Natal E Silva, Universidade Federal de Goiás, Goiânia, GO, 74605-170, Brazil.
Mol Biol Rep. 2020 Oct;47(10):7333-7340. doi: 10.1007/s11033-020-05772-1. Epub 2020 Sep 10.
Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.
登革热病毒和 Zika 病毒是虫媒黄病毒，在全球范围内导致数百万例感染。这两种病毒的共同传播使得血清学诊断变得困难，因为它们在病毒蛋白中的氨基酸高度相似。抗原是通过在感染恢复期的血清学检测中检测 IgG 抗体来区分这两种病毒的关键试剂之一。在这里，我们报告了使用杆状病毒表达载体系统在昆虫细胞中表达含有非保守和免疫显性氨基酸序列的登革热病毒（DENV）和 Zika 病毒（ZIKV）抗原。我们根据 E 蛋白（EDIII）的结构域 III 分析了先前报道的表位，并对最重要的黄病毒进行了多重比对，设计了 DENV 和 ZIKV 抗原。ZIKV 和 DENV 多表位基因设计为串联重复或缺失重复，由四或六甘氨酸接头隔开。生化分析显示抗原表达充分。然后，在蔗糖梯度中对获得的多表位抗原进行半纯化，并使用先前诊断为抗 DENV 和 -ZIKV IgG 抗体阳性的恢复期患者血清进行测试。最佳血清稀释度为 1:200，初步测试中的平均吸光度值表明多表位抗原已被人血清识别。基于 EDIII 区域的真核系统中多表位策略的这两种抗原的产生为区分 DENV 和 ZIKV 提供了抗原使用的概念验证。
