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Applied and Environmental Microbiology, October 2008, p. 6254-6262, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.00109-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of DegQ_Vh, a Serine Protease and a Protective Immunogen from a Pathogenic Vibrio harveyi Strain ^,

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, People's Republic of China,¹ Graduate University of the Chinese Academy of Sciences, Beijing 100049, People's Republic of China²

Received 14 January 2008/ Accepted 13 August 2008

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ_Vh), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ_Vh was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQ_Vh in T4 was modulated by temperature, possibly through the ^E-like factor. Enzymatic analyses demonstrated that the recombinant DegQ_Vh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQ_Vh protein were 50°C and pH 8.0. A vaccination study indicated that the purified recombinant DegQ_Vh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQ_Vh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQ_Vh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular β-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQ_Vh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ_Vh significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Published ahead of print on 22 August 2008.

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