Part:BBa_K3916004

RBD

Profile

Usage and Biology

BBa_K3916004 is the coding sequence of a kind of protein (RBD). Coronavirus pneumonia (Coronavirus disease 2019, COVID-19) infects the human body with pneumonia caused by the combination of S protein on its surface with angiotensin-converting enzyme 2 (ACE2) receptors. Mutations in the S1 protein in the S protein and its receptor binding domain (RBD) can lead to changes in viral infection capacity and may lead to immune escape. The RBD is also a significant neutralization determinant in the inactivated SARS-CoV vaccine because it induces potent NAbs that block SARS-CoV entry. In this project, we aimed to construct a plasmid to give the protein of RBD synthesized from B138 the ability to perform the protein purification and ELISA testing as well as to mimic the property held by the spike protein of the coronavirus.

Figure1 DNA gel electrophoresis..

Proof of function

3E8 Abolishes The Infectivity Of Multiple Pseudo-Typed Coronaviruses

Figure2 3E8 blocked the infection of ACE2-expressing cells by multiple pseudo-typed coronavirus constructs. ACE2-Fc and B38 were used as positive controls, and human IgG4 isotype was negative control. (A-G) 3E8 blocked the infection of ACE2-overexpressing HEK293 cells by different pseudo-typed coronavirus constructed with Env-defective HIV-1 and full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63. (H) The IC50 values of 3E8 in blocking pseudo-typed coronaviruses..

We next constructed pseudo-typed coronaviruses with full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63 (Fig. 2A-G). All pseudoviruses could infect HEK293F cells that ectopically express human ACE2, while SARS-CoV-2-D614G showed significantly enhanced infectivity when compared to the original SARS-CoV-2 (Fig. S2). Incubation with 3E8 fully abolished the infectivity of all pseudoviruses, with IC50 values at 0.1, 0.1, 0.07, 0.3, 0.08, 0.2 and 1.1 nM, respectively (Fig. 2H). In comparison, B38, a SARS-CoV-2 RBD-targeting antibody currently under clinical development, could only suppress the infectivity of SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7 and B.1.617, but not B.1.351, SARS-CoV or HCoV-NL63. The suppression of 3E8 was not only broader but also remarkably more efficacious and potent, as the IC50 values of 3E8 were hundreds of folds improved when compared to that of B38 (Fig. 2H). The ACE2-Fc fusion protein, a virus RBD-targeting molecule consisting of the extracellular domain of human ACE2 and the Fc region of human IgG1, showed broad but mediocre blocking ability on pseudoviruses. Our investigation indicated that 3E8 is potentially a powerful and broad-spectrum blocker on coronaviruses that are dependent on ACE2.

References

1.Assistant Secretary for Public Affairs (ASPA). (2021, June 28). Monoclonal Antibodies for High-Risk COVID-19 Positive Patients. combatCOVID.hhs.gov. 2. Boopathi, S., Poma, A. B., & Kolandaivel, P. (2021, June). Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. Journal of biomolecular structure & dynamics. 3. Centers for Disease Control and Prevention. (n.d.). Test for Current Infection. Centers for Disease Control and Prevention. 4. Huang, Y., Sun, H., Yu, H., Li, S., Zheng, Q., & Xia, N. (2020, December 28). Neutralizing antibodies against SARS-CoV-2: current understanding, challenge and perspective. Antibody therapeutics.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 417
  Illegal SapI.rc site found at 630