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United States Department of Agriculture

Agricultural Research Service

Research Project: ANAEROBIC MICROBIOLOGICAL PROCESSES IN ANIMAL MANURE MANAGEMENT Title: Engineering of the Human Commensal Bacteroides Ovatus for the in Situ Delivery of Immunomodulatory Proteins

Authors
item Hamady, Zaed - UNIV. OF LEEDS, UK
item Farrar, Mark - UNIV. OF LEEDS, UK
item Whitehead, Terence
item Lodge, J - UNIV. OF LEEDS, UK
item Carding, Simon - UNIV. OF LEEDS, UK

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 24, 2006
Publication Date: June 24, 2006
Citation: Hamady, Z.Z., Farrar, M.D., Whitehead, T.R., Lodge, J.P., Carding, S.R. 2006. Engineering of the human commensal Bacteroides ovatus for the in situ delivery of immunomodulatory proteins [abstract]. Reproduction Nutrition Development. 46(S64):0-18.

Technical Abstract: Soluble growth factors that can improve the intestinal barrier function such as keratinocyte growth factor (KGF) or oral tolerance such as transforming growth factor (TGF)-beta are of interest as potential therapeutic agents for inflammatory bowel disease. However, when administered orally as recombinant proteins they are unstable, and systemic administration increases the risk of unwanted side effects. Alternative means of delivery have been considered of which delivery via live microorganisms has shown real promise. The aim of this work was to genetically engineer the human commensal colonic bacterium, Bacteroides ovatus, to produce and secrete mammalian cytokines under the control of the xylanase promoter. The xylanase promoter was cloned and sequenced using an inverse-PCR approach. The coding sequence of the mature human cytokines TGF-beta or KGF was PCR amplified from cDNA and cloned downstream of the xylanase promoter in the Escherichia coli-Bacteroides suicide vector pBT2. This construct was introduced into B. ovatus by conjugation. Resulting transconjugants were tested for cytokine gene expression by reverse transcription-PCR and protein production by enzyme-linked immunosorbent assay (ELISA). Recombinant strains of B. ovatus were able to secrete human TGF-beta or KGF in a xylan-dependent manner. Cytokine expression was minimal in the absence of xylan. When xylan was added, cytokine expression and secretion increased and then reduced on removal of xylan from the culture media. Future work will assess the efficacy of these strains in the treatment and prevention of chemically induced colitis in mice.

Last Modified: 11/27/2014
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