Department of Biology, Shahid Chamran University of Ahvaz, Ahvaz, Khouzestan, Iran.
Department of Biology, Shahid Chamran University of Ahvaz, Ahvaz, Khouzestan, Iran.
Protein Expr Purif. 2023 Mar;203:106210. doi: 10.1016/j.pep.2022.106210. Epub 2022 Dec 4.
Many efforts have been made around the world to combat SARS-CoV-2. Among these are recombinant antibodies considered to be suitable as an alternative for some diagnostics/therapeutics. Based on their importance, this study aimed to investigate the expression, purification, and efficiency of a new potent recombinant scFv in the E. coli BL21 (DE3) system. The expression studies were performed after confirming the scFv cloning into the pET28a vector using specific PCRs. After comprehensive expression studies, a suitable strategy was adopted to extract and purify periplasmic proteins using Ni-NTA resin. Besides the purified scFv, the crude bacterial lysate was also used to develop a sandwich ELISA (S-ELISA) for the detection of SARS-CoV-2. The use of PCR, E. coli expression system, western blotting (WB), and S-ELISA confirmed the functionality of this potent scFv. Moreover, the crude bacterial lysate also showed good potential for detecting SARS-CoV-2. This could be decreasing the costs and ease its utilization for large-scale applications. The production of high-quality recombinant proteins is essential for humankind. Moreover, with attention to the more aggressive nature of SARS-CoV-2 than other coronaviruses, the development of an effective detection method is urgent. Based on our knowledge, this study is one of the limited investigations in two fields: (1) The production of anti-SARS-CoV-2 scFv using E. coli [as a cheap heterologous host] in relatively high amounts and with good stability, and (2) Designing a sensitive S-ELISA for its detection. It may also be utilized as potent therapeutics after further investigations.
世界各地已经做出了许多努力来对抗 SARS-CoV-2。其中包括被认为适合替代某些诊断/治疗方法的重组抗体。基于其重要性，本研究旨在调查新的强效重组 scFv 在大肠杆菌 BL21 (DE3) 系统中的表达、纯化和效率。在使用特定 PCR 确认 scFv 克隆到 pET28a 载体后，进行了表达研究。经过全面的表达研究，采用了一种合适的策略，使用 Ni-NTA 树脂提取和纯化周质蛋白。除了纯化的 scFv 外，还使用粗菌裂解物开发了用于检测 SARS-CoV-2 的夹心 ELISA (S-ELISA)。PCR、大肠杆菌表达系统、Western blot (WB) 和 S-ELISA 的使用证实了这种强效 scFv 的功能。此外，粗菌裂解物也显示出检测 SARS-CoV-2 的良好潜力。这可以降低成本并使其更易于大规模应用。生产高质量的重组蛋白对人类至关重要。此外，鉴于 SARS-CoV-2 比其他冠状病毒具有更强的攻击性，因此迫切需要开发有效的检测方法。根据我们的知识，这项研究是在两个领域进行的有限调查之一：(1) 使用大肠杆菌 [作为廉价的异源宿主] 以相对较高的量和良好的稳定性生产抗 SARS-CoV-2 scFv，以及 (2) 设计用于其检测的敏感 S-ELISA。在进一步研究后，它也可能被用作有效的治疗方法。
