Abstract

The receptor binding domain (RBD) of SARS-CoV-2 is located in the C-terminal of S1 subunit of the spike (S) protein which is responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The DNA encoding the SARS-CoV-2 RBD was inserted into pET-28a (+) to construct expression plasmid pET-28a (+)/RBD. The desired RBD protein was produced in E. coli Rosetta (DE) and purified by a Ni-NTA column. The recombinant RBD was analyzed by SDS-PAGE and Western blot. The flow cytometry analysis indicated that the recombinant RBD is capable of binding to human ACE2 (hACE2) in the ACE2-overexpressed HEK293A-hACE2 cells. Our results demonstrated that recombinant RBD expressed in E. coli Rosetta (DE) strain has bioactivities and can be used as an antigen for diagnosis and as a tool for the development of novel anti-viral drugs against SASR-CoV-2.

摘要

SARS-CoV-2 的受体结合域 (RBD) 位于刺突 (S) 蛋白 S1 亚基的 C 末端，负责识别和结合血管紧张素转换酶 2 (ACE2) 受体。将编码 SARS-CoV-2 RBD 的 DNA 插入 pET-28a (+) 以构建表达质粒 pET-28a (+)/RBD。所需的 RBD 蛋白在大肠杆菌Rosetta (DE) 中产生并通过 Ni-NTA 柱纯化。通过SDS-PAGE和Western印迹分析重组RBD。流式细胞仪分析表明，重组RBD能够与ACE2过表达的HEK293A-hACE2细胞中的人ACE2（hACE2）结合。我们的结果表明，在大肠杆菌中表达的重组 RBDRosetta (DE) 菌株具有生物活性，可用作诊断抗原和开发针对 SASR-CoV-2 的新型抗病毒药物的工具。