免疫调节性PanDR和SARS-CoV-2受体结合域抗原在细菌宿主细胞中的表达及免疫测定评估
Expression and immunoassay assessment of immunomodulatory PanDR and SARS-CoV-2 receptor-binding domain antigen in bacteria host cell.
摘要(中文译文)
在低收入和中等收入国家（LMICs），生物制造技术能力的差距和不完善的基础设施限制了疫苗研发和生物制造的进展。肽亚单位抗原为生产针对SARS-CoV-2及其他新发病毒性疾病的经济有效、安全且具有保护性的疫苗提供了一种有前景的解决方案。SARS-CoV-2刺突蛋白的受体结合域（RBD）通过与人血管紧张素转换酶2（ACE2）受体相互作用介导病毒进入，是构建和加强本地能力的合适模型抗原。本研究旨在利用细菌表达系统表达并验证重组SARS-CoV-2 RBD蛋白作为疫苗候选物。在本研究中，RBD序列与PanDR表位序列（一种免疫刺激剂）连接，随后进行密码子优化以适用于细菌表达。利用计算机模拟工具评估该蛋白序列的理化参数以评价其适用性。该序列经化学合成后克隆至pMAL-c6T表达载体，随后转化至大肠杆菌BL21-DE3细胞中。通过IPTG诱导在Luria-Bertani营养培养基中表达3小时，然后利用亲和层析进行纯化。SDS-PAGE显示在约74.5 kDa的预期分子量处出现明显条带，对应MBP-RBD融合蛋白，表明该重组蛋白在细菌表达系统中成功表达。Western blot分析通过抗MBP抗体的特异性识别进一步证实了该蛋白的身份。结果表明，修饰后的SARS-CoV-2受体结合域蛋白已成功合成、克隆、表达并验证，可用于进一步的免疫学评价。
Abstract
The gap in technical capacity and inadequate infrastructure for biomanufacturing limits progress in vaccine development and biomanufacturing in low- and middle-income countries (LMICs). Peptide subunit antigens offer a promising solution to producing cost-effective, safe and protective vaccines for SARS-CoV-2 and other emerging viral diseases. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates viral entry through interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor, represents a suitable model antigen for building and strengthening local capacity. The aim of this study is to express and validate a recombinant SARS-CoV-2 RBD protein as a vaccine candidate using bacterial expression systems. In this study the RBD sequence is linked to PanDR epitope sequence, an immunostimulant' then codon optimized for bacterial expression. The physicochemical parameter of the protein sequence was assessed using in-silico tools to assess its suitability. The sequence was synthesized chemically, and cloned into a pMAL-c6T expression vector, then transformed into Escherichia coli BL21-DE3 cells. The protein was expressed by IPTG induction for 3 h in Luria-Bertani nutrient media and then purified using affinity chromatography. The recombinant protein was successfully expressed in a bacterial expression system as revealed by a distinct band at the expected molecular weight of approximately 74.5 kDa, on SDS-PAGE corresponding to the MBP-RBD fusion protein. Western blot analysis further confirmed the identity of the protein through specific recognition by anti-MBP antibody. The results show that the modified receptor binding domain protein of SARS-CoV-2 was successfully synthesized, cloned, expressed and validated for further immunological evaluation.
如何引用
AMA
Rukayat Olajumoke Onilegbale, Gbemisola Morounke Saibu, Habeeb Adebodun Bankole, Rebecca Oziohu Omosimua, Muinah Adenike Fowora, Ayorinde Babatunde James. Expression and immunoassay assessment of immunomodulatory PanDR and SARS-CoV-2 receptor-binding domain antigen in bacteria host cell.. Protein expression and purification. 2027; doi:10.1016/j.pep.2026.106982.
APA
Rukayat Olajumoke Onilegbale, Gbemisola Morounke Saibu, Habeeb Adebodun Bankole, Rebecca Oziohu Omosimua, Muinah Adenike Fowora, Ayorinde Babatunde James (2027). Expression and immunoassay assessment of immunomodulatory PanDR and SARS-CoV-2 receptor-binding domain antigen in bacteria host cell.. Protein expression and purification. https://doi.org/10.1016/j.pep.2026.106982
GB/T 7714
Rukayat Olajumoke Onilegbale, Gbemisola Morounke Saibu, Habeeb Adebodun Bankole, Rebecca Oziohu Omosimua, Muinah Adenike Fowora, Ayorinde Babatunde James. Expression and immunoassay assessment of immunomodulatory PanDR and SARS-CoV-2 receptor-binding domain antigen in bacteria host cell.[J]. Protein expression and purification, 2027 doi:10.1016/j.pep.2026.106982.
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