Tabriz University of Medical Sciences BioImpacts 2228-5652 15 1 2025 01 19 Performance of protein N linear epitopes in serodiagnosis of COVID-19 infection 30232 30232 10.34172/bi.30232 EN Sahar Farajnia https://orcid.org/0009-0001-6622-9014 Nazli Khajenasiri Safar Farajnia https://orcid.org/0000-0002-6087-9147 Farzin Seyrafi Nasim Bakhtiyari Journal Article 10.34172/bi.30232 2023 12 20 2024 02 28 Introduction: Despite the efforts to contain the spread of COVID-19, the virus remains in circulation, posing a considerable risk to populations across the globe. Hence, rapid and early detection of this infection is essential for effective disease control. The nucleocapsid (N) protein of the virus serves as a primary target for antibody response during CoV2 infections, making it a potential candidate for COVID-19 detection. This study aims to prepare and evaluate the linear epitopes of the N protein for serodiagnosis of COVID-19 infection. Methods: The linear epitope of the N protein gene was identified using ABCpred, BCpred, and IEDB. These epitopes were subsequently amplified by RT-PCR, cloned, and expressed in soluble form in the E. coli BL21 strain. The recombinant protein was purified using the Ni-NTA column. The reactivity of purified N protein with sera from SARS-CoV-2 patients was analyzed using an ELISA assay. Results: Sequencing analysis demonstrated the successful cloning of the linear epitopes of the N protein into the PET-28a vector, along with an n-terminal His-tag fusion. The recombinant protein was produced in E. coli BL21 and purified with a Ni-NTA column. The analysis demonstrated that the N protein linear epitopes were expressed in a soluble form and appeared as a 50 kDa band in the SDS-PAGE. Examination for the reactivity of the purified N protein with the COVID-19 patient’s sera by ELISA revealed that the N protein recognizes the infection with high sensitivity and specificity. Conclusion: The results of this study indicated that linear epitopes of the SARS-CoV-2 N protein are highly immunogenic and could be exploited for serodiagnosis of infection in patients suspected of COVID-19 infection.