Date

2022-9-15

Author

Keser, Duygu

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COVID-19 is an infectious disease caused by SARS-CoV-2. The virus was first detected in Wuhan, China in late 2019, and the outbreak was declared a pandemic in January 2020 by WHO, and continues to spread worldwide. As of July 2022, more than 575 million confirmed cases have been detected all over the world, and more than 6 million people died from the disease. One of the most important public health measures in combating the spread of infectious diseases is vaccination. Despite the existence of rapidly developed and administered vaccines in the later stages of the COVID-19 pandemic, there is still a need to develop more effective and safe vaccines against this pathogen. As a result, there is an urgent need to examine proteins with high immunogenicity that can be employed in novel vaccines. The genome of SARS-CoV-2 encodes four structural proteins and other accessory or nonstructural proteins. The present study is on the evaluation of the immunogenicity of the S1 and S2 region protein fragments in the Spike protein and the whole nucleocapsid protein for the formulation of a recombinant subunit vaccine against SARS-CoV-2. S1, S2 gene fragments and the N gene were amplified by PCR and cloned into the pGEM®-T Easy vector. The genes were then inserted into the pET-28a (+) vector and their gene expression was achieved in Escherichia coli BL21(DE3) cells. Recombinant proteins were purified by His-tag affinity chromatography. Western blot analyzes were performed with monoclonal antibodies and immunized mice sera. Serum-specific IgG levels were measured by the ELISA method, and the increased level of total antibodies to all three antigens showed that a robust humoral response was developed in the immunized group of mice. Moreover, in the analyzes depend on the ELISA results, a significant increase was observed in the antigen-specific IgG2a titers, which indicate the cellular response, in the immunized group compared to the control group. The increase in IFN-gamma levels observed as a result of the performed cytokine ELISAs indicated a strong cellular response to recombinant antigens and, in addition to the antibody response, indicates that these three antigens are suitable vaccine antigen candidates to combat the COVID-19 pandemic.

Subject Keywords

SARS-CoV-2, COVID-19, Recombinant protein vaccine, Immune response, Spike, Nucleocapsid

URI

https://hdl.handle.net/11511/99457

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Graduate School of Natural and Applied Sciences, Thesis