Browsing by Author "Edwards, Andrew"

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    Mapping the Major Epitope(s) of the Glycoprotein of Ebolavirus with QB Phage Display System
    (2023-02) Edwards, Andrew; Waffo, Alain B.; Elmendorf, Jeffrey S.; Quilliam, Lawrence A.
    The main goal of the research was to produce and analyze Qβ phage virions containing various segments of the EBOV glycoprotein fused to its A1 the readthrough minor coat protein. The recombinant phages would then be utilized to analyze their antigenicity and to map the major epitope(s) of the glycoprotein determinants. This research study represents a proof of concept and will serve as a guide as to how to produce recombinant phages bearing large antigenic peptide segments of a viral protein and initiate its analysis. To prepare the recombinant plasmids, separately, the gene segments of the glycoprotein determinants were designed with overlapping fragments, fused with the A1 gene and amplified by PCR. The obtained gene fragments were separately purified, digested and ligated with a modified phage display plasmid vector pBR322 containing a full copy of cDNA genome of the Qβ phage. The resulting ligation was used to transform E. coli DH5α competent cells. Colonies were picked, grown overnight, plasmids purified, analyzed using restriction enzymes, and confirmed via Sanger sequencing. Positively sequenced plasmid clones were used to retransform E. coli HB101 for phages production. The phage titer was 105 pfu/ml, lower than that typically achieved with wildtype phages. The recovery and resuspension of the phages from PEG/NaCl was further scaled to a titer of 1012 pfu/ml, followed by precipitation, and RNA isolation. The recombinant RNA was used to obtain cDNA for PCR amplification. The amplified cDNA was analyzed by agarose gel electrophoresis and sequenced to confirm the presence, position, and orientation of the glycoprotein segment within the recombinant phages. We were able to successfully produce recombinant phages harboring gene segments of the Ebolavirus glycoprotein fused to the Qβ phages A1 coat protein. All the phages with the glycoprotein determinants will be analyzed for their antigenicity using specific glycoprotein antibody once they are available. Additionally, our panning methodology will be used to determine the segment(s) containing the major epitopes. The segment will be confirmed using blotting and agarose double diffusion technique. This work can be extended to identify antibody epitopes in other RNA viruses and as a point of care for major infectious diseases.