Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity – an integrated experimental and bioinformatic approach.

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity – an integrated experimental and bioinformatic approach.

Publication date: Jan 17, 2024

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence. Communicated by Ramaswamy H. Sarma.

Concepts Keywords
Bioinformatic COVID-19
Bl21 SARS-CoV-2 ELISA
Pandemic Shuffle T7 expression
Pro TRX-tag fusion protein
Vaccine

Semantics

Type Source Name
disease MESH COVID-19 pandemic
disease VO effective
drug DRUGBANK Glycine
drug DRUGBANK Sodium lauryl sulfate
disease IDO assay
disease VO vaccine

Original Article

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