SARS_CoV2 RBD gene transcription cannot be driven by CMV promoter

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• The SARS-CoV-2 Receptor Binding Domain (RBD) is crucial for viral entry and a key target for therapeutics.
• Efficient expression of native conformational RBD protein is vital for drug and vaccine development against COVID-19.
• The Cytomegalovirus (CMV) promoter is commonly used for in vitro gene expression.

Purpose of the Study:

• To investigate the efficacy of the CMV promoter for expressing SARS-CoV-2 RBD protein.
• To identify alternative promoters capable of driving high-level SARS-CoV-2 RBD expression.
• To provide insights for optimizing RBD protein production for COVID-19 research.

Main Methods:

• Transfection of recombinant plasmids containing SARS-CoV-2 RBD gene under the control of different promoters into cells.
• Detection of RBD-specific mRNA using molecular assays.
• Assessment of CMV promoter activity in the presence of the SARS-CoV-2 RBD construct.

Main Results:

• The CMV promoter failed to drive SARS-CoV-2 RBD expression, with no detectable RBD-specific mRNA.
• The pCMV-Tag2B-SARS_CoV2_RBD construct was found to trans-inactivate the CMV promoter.
• Both the Chicken β-actin promoter and the Vaccinia virus M/L promoter (pSYN) demonstrated high-level expression of SARS-CoV-2 RBD.

Conclusions:

• The CMV promoter is unsuitable for expressing SARS-CoV-2 RBD protein.
• Chicken β-actin and Vaccinia virus M/L promoters are effective alternatives for SARS-CoV-2 RBD expression.
• These findings offer valuable guidance for future research involving SARS-CoV-2 RBD protein exploration and therapeutic development.