Abstract：
The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.
摘要：
严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）核衣壳蛋白（Ncap）的基本生物学，自2019年底Covid19大流行爆发以来，其在诊断测定中的应用及其作为疫苗成分的潜在应用受到了相当大的关注。在这里，我们报告了可溶性的可扩展表达和纯化，免疫活性,大肠杆菌中的SARS-CoV-2Ncap。将编码原始Ncap序列和具有N末端6His亲和标签（序列MHHHHHHHHHG）的四种常见变体的密码子优化合成基因克隆到诱导型表达载体中，该载体携带受lac操纵子结合位点控制的调节噬菌体T5合成启动子。构建体用于表达Ncap蛋白,并且开发了允许以超过200mg/升培养基的产量有效生产纯化的Ncap的方案。将这些蛋白质用于ELISA测定中以允许比较它们对人血清的应答。我们的结果表明，6His标记和未标记的原始Ncap蛋白之间没有可检测到的差异，但血清对其他Ncap分离株的敏感性可能略有下降。
