Date of Award

Summer 8-12-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Sidney A. Crow, Jr.

Second Advisor

George Pierce

Third Advisor

Eric Gilbert

Abstract

The protein monomer of bacterial flagella, FliC, is known to stimulate human innate immunity through activation of Toll-like receptor five. Linking native Salmonella FliC with various antigens has demonstrated an increased immune response as compared to single antigen presentation. To drastically reduce production time and allow for a more cost effective recombinant vaccine adjuvant, a synthetic construct was created that enables genetic linkage of FliC to other known antigens. The construct contains the necessary components for immune system stimulation while the non-essential regions were replaced with commonly used restriction enzyme recognition sites to aid in ligation with other antigens and cloning into various expression vectors and hosts. After synthesis in the inducible expression vector pJ404, the construct was transformed into competent BL21 E. coli and expression was confirmed through SDS-PAGE, Western blot, and MALDI MS/MS. The cells were adapted to fermentation media and re-screened for expression, and upon confirmation a 20-liter fermentation was conducted. The resulting samples were analyzed for expression within the insoluble and soluble cellular fractions to further optimize fermentation conditions. Once purified, this synthetic FliC will serve as a platform technology for the standardized co-expression of the TRL5 activator with a variety of antigens in both prokaryotic and eukaryotic systems.

DOI

https://doi.org/10.57709/5812212

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