Date of Award

12-14-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. George Pierce

Second Advisor

Dr. Sidney Crow

Third Advisor

Dr. Eric Gilbert

Abstract

Currently, there is no vaccine for Shigellosis. The Shiga-like toxoid 2, by itself, is poorly immunogenic. Flagellin based fusion proteins, serving as toll like receptor agonists, have shown both activation of innate immunity and an increase in adaptive immune responses from normally poorly immunogenic microenvironments. A synthetic flagellin based construct was designed to allow the insertion of detoxified antigen sequences, allowing multivalent expression and antigen presentation. Specifically, subunit B of shiga-like toxin 2, an Escherichia coli O157 verocytotoxin, was chosen for co-expression alongside the construct for antigen specific presentation. The fusion protein, FliC-Stx2B, contains the innate activating synthetic FliC construct along with the genetically modified Stx2B toxoid.

FliC-Stx2B amplicon DNA was produced through PCR while removing the associated signal peptide motif to prevent translocation and increase expression. Additionally, a hexa-histidine affinity tag was incorporated downstream of the amplicon to further aid in purification. Positive clones were induced with varying amounts of IPTG to determine solubility of expressed protein. A detergent wash method, using heavy detergents, was then used to resolubilize & isolate FliC-Stx2B from cell culture pellets. Purification was conducted using ÄKTA® FPLC systems utilizing nickel column affinity. FliC-Stx2B was then refolded while on-column to revert the fusion protein to an intrinsic state, then eluted. Biological activity was determined using Western Blot, ELISA, & TLR5 assays.

Over-expressed FliC-Stx2B was primarily found in insoluble fractions during culture induction studies. Non-desired proteins were removed from the induced insoluble cell pellet fractions during resolubilization, while also isolating FliC-Stx2B. The significant reduction in non-desired protein simplifies FliC-Stx2B purification further due to the addition of the hexa-histidine tag, allowing for nickel column affinity. Purification yields for FliC-Stx2B is reported between 45-70% across varying on-column incubation time points. Binding of anti-Stx2B mAbs, seen in both Western blot and indirect-ELISA assay, confirms that FliC-Stx2B biological activity is maintained following both insoluble protein resolubilization & purification. Preliminary TLR5 activity is seen in purified refolded FliC-Stx2B. These findings indicate that FliC-Stx2B retains its biological activity post-purification and could be a potential vaccine candidate.

DOI

https://doi.org/10.57709/11206691

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