Date of Award

Summer 8-8-2022

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Daniel Nichols, Ph.D.

Committee Member

Angela Klaus, Ph.D.

Committee Member

Cosimo Antonacci, Ph.D.

Keywords

SARS-CoV-2 S1, Fc Fusion, pFUSE, Spike Protein, IgG2a

Abstract

Despite the availability of vaccines and antiviral medication, SARS-CoV-2 still presents a significant threat to public health. New variants of SARS-CoV-2 continue to emerge. As a result, the need to develop and evaluate new vaccinations strategies is critical. In this thesis the S1 region (amino acid residues 14Q-685R) containing the receptor binding domain of the SARS-CoV-2 spike protein was fused to the Fc region of IgG2a mouse antibody. A polymerase chain reaction was done to amplify the segment of interest, in parallel to the digestion of the vector pFUSE-mIgG2a-Fc2. The experimental cloning was done by ligating the two segments together. To express and purify SARS-CoV-2 S1 14Q-685R/pFUSE, Chinese hamster ovaries cells were transfected with the plasmids and plated down with varying antibiotic concentrations of Zeocin. The antibiotics were used to select for the cells that took up the plasmids, since the pFUSE vector contain Zeocin resistant marker. Finally, the expression of the SARS-CoV-2 S1 14Q-685R/pFUSE was confirmed via immunoblotting. The Fc fused spike protein of the virus was precipitated using the addition of ammonium sulfate into the media. Furthermore, once enough concentration has been collected the samples will be passed through Pierce™ Protein G Chromatography columns to collect the IgG fused proteins which will be used for future studies to evaluate immune responses in mice.

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