Darestanifarahani Mahsa, Mahmoudi Faezeh, Mohammadi Ali, Lotfi Ehsan, Bahrami Bahar, Shajari Samira, Hamzehlooy Fatemeh, Karamali Fatemeh, Barati Mahmood, Jafari Davod
Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
Mol Biotechnol. 2025 Apr 7. doi: 10.1007/s12033-025-01424-6.
This study aimed to produce a recombinant N protein to develop an enzyme-linked immunosorbent assay (ELISA). A recombinant pET28a vector was constructed, and E. coli BL 21 was transformed for recombinant protein expression. After SDS-PAGE analysis, we approved recombinant N protein expression and purification using western and dot-blotting tests. In ELISA setup tests, 2.5 µg/ml of N protein and 1:100 serum dilution was determined as optimum concentrations for anti-N IgG antibody. In validation tests, 47 out of 51 negative and 46 out of 51 positive samples were determined negative and positive, respectively, using our developed ELISA. According to the Receiver Operating Characteristic (ROC) curve analysis based on the Youden index, the sensitivity and specificity of the developed test were 92% and 90.38%, respectively. An ELISA test with the recombinant SARS-CoV-2 N protein was developed with high specificity and sensitivity for the clinical diagnosis of SARS-CoV-2 infection.
本研究旨在制备重组N蛋白以开发酶联免疫吸附测定（ELISA）。构建了重组pET28a载体，并转化大肠杆菌BL 21用于重组蛋白表达。经过SDS-PAGE分析后，我们通过western和斑点印迹试验证实了重组N蛋白的表达和纯化。在ELISA设置试验中，确定2.5 µg/ml的N蛋白和1:100的血清稀释度为抗N IgG抗体的最佳浓度。在验证试验中，使用我们开发的ELISA，51份阴性样本中的47份和51份阳性样本中的46份分别被判定为阴性和阳性。根据基于约登指数的受试者工作特征（ROC）曲线分析，所开发检测方法的灵敏度和特异性分别为92%和90.38%。开发了一种使用重组SARS-CoV-2 N蛋白的ELISA检测方法，对SARS-CoV-2感染的临床诊断具有高特异性和灵敏度。
