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

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.

在新型冠状病毒肺炎（COVID-19）大流行期间，开发高效的诊断检测方法对于遏制严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的传播至关重要。多结构域融合蛋白是检测SARS-CoV-2抗体的极具潜力的策略。本研究中，我们设计了一款名为CoV2-Pro的抗原，其包含两段分别来源于SARS-CoV-2奥密克戎（Omicron）和德尔塔（Delta）变异株的受体结合域（Receptor Binding Domain, RBD），以及一段核蛋白的羧基末端结构域（C-terminal Domain, CTD），序列排布为RBD-RBD-N，并通过超强柔性甘氨酸接头进行连接。我们评估了大肠杆菌（Escherichia coli）Shuffle T7与BL21 (DE3)菌株用于表达CoV2-Pro的适配性。此外，我们首先通过生物信息学分析手段解析了CoV2-Pro的三级结构。我们将CoV2-Pro的编码序列克隆至pET-32b (+)载体中，以在大肠杆菌Shuffle T7与BL21 (DE3)菌株中实现表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与蛋白质印迹法验证了目标蛋白的表达与折叠状态。我们通过镍柱亲和层析（Ni-NTA亲和层析）纯化得到了CoV2-Pro-TRX融合蛋白。我们采用斑点印迹法（Dot Blot）分析了CoV2-Pro的抗原特性。我们开展了分子对接模拟实验，以评估CoV2-Pro与LY-COV555（巴尼韦单抗，Bamlanivimab）单克隆抗体的结合亲和力。我们还通过分子动力学模拟分析了CoV2-Pro结构的稳定性。生物信息学与实验研究结果均证实，CoV2-Pro具备稳定的三维构象结构。CoV2-Pro可与SARS-CoV-2抗体特异性结合，验证了其抗原结构的正确性。我们有充分依据认定，CoV2-Pro是开发酶联免疫吸附测定（ELISA）检测方法的理想抗原，可用于COVID-19大流行期间的精准诊断与严格的疫苗效力评估。