Samiei-Abianeh Hossein, Nazarian Shahram, Kordbacheh Emad, Felegary Alireza
Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.
Bioimpacts. 2024 Nov 4;15:30520. doi: 10.34172/bi.30520. eCollection 2025.
The SARS-CoV-2 pandemic necessitates effective therapeutic solutions. The receptor-binding motif (RBM) is a subdomain of the spike protein's receptor-binding domain (RBD) and is critical for facilitating the binding of SARS-CoV-2 to the human ACE2 receptor. This study investigates the use of the receptor-binding motif (RBM) domain as an immunogen to produce potent neutralizing antibodies against SARS-CoV-2.
The RBM gene was codon-optimized and cloned into the pET17b vector for expression in BL21 (DE3) cells, induced with 1 mM IPTG. The recombinant RBM protein was purified using Ni-NTA affinity chromatography. After validating the recombinant RBM by Western blotting with anti-His tag antibodies, BALB/c mice were immunized with 20 µg of the purified RBM protein. Anti-RBM IgG was subsequently purified using protein G resin, and its neutralizing capacity was assessed using the Pishtaz Teb Zaman Neutralization Assay Kit.
The recombinant RBM protein, with a molecular weight of 10 kDa, was expressed as inclusion bodies. the typical yield of purification was 27 mg/L of bacterial culture. The neutralization test demonstrated a concentration of 36 µg/mL of neutralizing antibodies in the immunized serum, preventing the spike protein from binding to ACE2.
Our study demonstrated that anti-RBM antibodies exhibited neutralization effects on SARS-CoV-2. These findings provide evidence for the development of a vaccine candidate through the induction of antibodies against the RBM, necessitating further studies with adjuvants suitable for human use to evaluate its potential for human vaccination.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）大流行需要有效的治疗解决方案。受体结合基序（RBM）是刺突蛋白受体结合域（RBD）的一个亚结构域，对于促进SARS-CoV-2与人血管紧张素转换酶2（ACE2）受体的结合至关重要。本研究调查了使用受体结合基序（RBM）结构域作为免疫原以产生针对SARS-CoV-2的强效中和抗体。
对RBM基因进行密码子优化，并克隆到pET17b载体中，以便在BL21（DE3）细胞中表达，用1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导。重组RBM蛋白使用镍-次氮基三乙酸（Ni-NTA）亲和层析法纯化。在用抗组氨酸标签抗体进行蛋白质印迹法验证重组RBM后，用20 μg纯化的RBM蛋白免疫BALB/c小鼠。随后使用蛋白G树脂纯化抗RBM IgG，并使用Pishtaz Teb Zaman中和检测试剂盒评估其中和能力。
分子量为10 kDa的重组RBM蛋白以包涵体形式表达。纯化的典型产量为每升细菌培养物27 mg。中和试验表明，免疫血清中中和抗体的浓度为36 μg/mL，可阻止刺突蛋白与ACE2结合。
我们的研究表明，抗RBM抗体对SARS-CoV-2具有中和作用。这些发现为通过诱导针对RBM的抗体来开发候选疫苗提供了证据，有必要进一步研究适合人类使用的佐剂，以评估其用于人类疫苗接种的潜力。
