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Infection and Immunity, May 2009, p. 1817-1826, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01301-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of Non-Antibiotic-Resistant, Chromosomally Based, Constitutive and Inducible Expression Systems for aroA-Attenuated Salmonella enterica Serovar Typhimurium {triangledown}

Jake N. Matic,1,2 Tamsin D. Terry,3 David Van Bockel,1,2 Tracy Maddocks,1 David Tinworth,4 Michael P. Jennings,3 Steven P. Djordjevic,2 and Mark J. Walker1*

School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia,1 Elizabeth Macarthur Agricultural Institute, Menangle, NSW, Australia,2 School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia,3 Bioproperties, Melbourne, Victoria, Australia4

Received 24 October 2008/ Returned for modification 18 December 2008/ Accepted 9 February 2009

Live-vaccine delivery systems expressing two model antigens from Mycoplasma hyopneumoniae, F2P97 (Adh) and NrdF, were constructed using Salmonella enterica serovar Typhimurium aroA (STM-1), and immunogenicity in mice was evaluated. Recombinant plasmid-based expression (PBE) and chromosomally based expression (CBE) systems were constructed. The PBE system was formed by cloning both antigen genes into pJLA507 to create an operon downstream of temperature-inducible promoters. Constitutive CBE was achieved using a promoter-trapping technique whereby the promoterless operon was stably integrated into the chromosome of STM-1, and the expression of antigens was assessed. The chromosomal position of the operon was mapped in four clones. Inducible CBE was obtained by using the in vivo-induced sspA promoter and recombining the expression construct into aroD. Dual expression of the antigens was detected in all systems, with PBE producing much larger quantities of both antigens. The stability of antigen expression after in vivo passage was 100% for all CBE strains recovered. PBE and CBE strains were selected for comparison in a vaccination trial. The vaccine strains were delivered orally into mice, and significant systemic immunoglobulin M (IgM) and IgG responses against both antigens were detected among all CBE groups. No significant immune response was detected using PBE strains. Expression of recombinant antigens in S. enterica serovar Typhimurium aroA from chromosomally located strong promoters without the use of antibiotic resistance markers is a reliable and effective method of inducing a significant immune response.

* Corresponding author. Mailing address: School of Biological Sciences, University of Wollongong, NSW, 2522, Australia. Phone: 61-2-42213439. Fax: 61-2-42214135. E-mail: mwalker{at}uow.edu.au

{triangledown} Published ahead of print on 17 February 2009.

Editor: F. C. Fang

Infection and Immunity, May 2009, p. 1817-1826, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01301-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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