Abstract：
Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host\'s cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.
摘要：
昆虫细胞-杆状病毒表达载体系统是目前最为成熟的生物制品生产平台之一,它在COVID-19紧急情况下发挥着重要作用，提供用于治疗的重组蛋白，诊断,和预防。SARS-CoV-2感染是由刺突糖蛋白三聚体通过其受体结合域（RBD）与宿主细胞受体相互作用介导的。由于许多应用程序都需要RBD，在大流行的背景下,重要的是应对产生大量重组RBD(rRBD)的挑战。出于这个原因,在本研究中，我们开发了一种基于Sf9昆虫细胞的工艺来提高rRBD产量。从感染细胞的上清液中回收rRBD，并通过金属离子亲和层析容易纯化，产率82%,纯度高于95%。在新型嵌合启动子（polh-pSeL）下表达，纯化后的rRBD收率为21.1±3.7mg/L，这是Sf9细胞系中描述的最高性能。最后,rRBD成功用于检测COVID-19血清样品中的特异性抗体。本文所述的有效策略具有在Sf9细胞系中产生用于诊断目的的高质量rRBD的潜力。
