Characterization of DegQVh, a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain.
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 sigma(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 degrees 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 beta-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.
Zhang
Wei-wei
WW
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, People's Republic of China.
Sun
Kun
K
Cheng
Shuang
S
Sun
Li
L
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005393:Fish Diseases / Q000382:microbiology*; D005432:Flounder; D015870:Gene Expression; D005816:Genetic Complementation Test; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D016019:Survival Analysis; D014733:Vibrio / Q000201:enzymology* / Q000276:immunology / Q000302:isolation & purification; D014735:Vibrio Infections / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary*
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D012697:Serine Endopeptidases