Targeted inhibition of Escherichia coli O157:H7 using VHH-based recombinant protein and antibody-drug conjugate

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dc.contributor.advisor Huang, Kang
dc.contributor.author Zhang, Hanxiaoya
dc.date.accessioned 2022-07-27T01:05:54Z
dc.date.available 2022-07-27T01:05:54Z
dc.date.issued 2022 en
dc.identifier.uri https://hdl.handle.net/2292/60597
dc.description Full Text is available to authenticated members of The University of Auckland only. en
dc.description.abstract The increasing occurrence of Escherichia coli (E. coli) O157:H7 outbreaks has been dealing significant damage to human health and the global economy. The negative side effects of conventional antibiotic treatments and the emergence of multidrug-resistant E. coli strains are urging for a new solution for better prevention and treatment of E. coli O157:H7 infections. Originally designed for cancer treatment, targeted therapies have shown great potential for the prevention and treatment of E. coli O157:H7 infections. It is possible to produce recombinant proteins and antibody-drug conjugates (ADCs) that contain an antibody portion and a drug portion to selectively attack pathogenic cells in a complex environment, providing pathogen-specific inhibitory effects. In this project, an antibacterial recombinant protein and an ADC were developed based on an anti-E. coli O157:H7 heavy chain variable domain (VHH) antibody. Positive phage clones were first obtained through affinity panning of a naïve phage display library using E. coli O157:H7 surface protein peptide fragments as coating antigens. When tested against live cells of E. coli O157:H7 ATCC 43888, positive phage clone 10E and 17O showed limits of detection (LODs) of 3.40 × 105 CFU/mL and 1.25 × 106 CFU/mL. The VHH sequences were then extracted from 10E and 17O phage clones and inserted into suitable plasmid vectors for prokaryotic gene expression. The resulting GST-tagged VHH antibodies, including 10E-GST and 17O-GST, achieved LODs of 4.15 × 106 CFU/mL and 1.31 × 107 CFU/mL against E. coli O157:H7 ATCC 43888, respectively. Afterward, the best performing 10E-GST was fused with a novel antimicrobial peptide (AMP). AA139 and conjugated with chlorogenic acid that is identified as an antibacterial phytochemical. The resulting recombinant protein 10E-AMP-GST and ADC 10E-GSTCGA exhibited good antibacterial properties against E. coli O157:H7, the minimum inhibitory concentrations being 0.13 mg/mL and 0.14 mg/mL, respectively. However, no significant binding affinity was shown by either the recombinant protein or the ADC. Although the two antibacterial agents demonstrated prevention and treatment effects during the infection assay, no selective inhibition was observed when E. coli O157:H7 was mixed with S. typhimurium. The results in this project will be valuable for future studies on the production of VHH-based recombinant proteins and ADCs.
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof Masters Thesis - University of Auckland en
dc.relation.isreferencedby UoA en
dc.rights Restricted Item. Full Text is available to authenticated members of The University of Auckland only. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/3.0/nz/
dc.title Targeted inhibition of Escherichia coli O157:H7 using VHH-based recombinant protein and antibody-drug conjugate
dc.type Thesis en
thesis.degree.discipline Food Science
thesis.degree.grantor The University of Auckland en
thesis.degree.level Masters en
dc.date.updated 2022-06-27T00:30:18Z
dc.rights.holder Copyright: the author en


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