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Identification and evaluation as a DNA vaccine candidate of a virulence-associated serine protease from a pathogenic Vibrio parahaemolyticus isolate

Liu, Rui, Chen, Jixiang, Li, Kesheng, Zhang, Xiaohua
Fish & shellfish immunology 2011 v.30 no.6 pp. 1241-1248
Danio rerio, Escherichia coli, Scophthalmus maximus, Vibrio parahaemolyticus, Western blotting, agar, amino acids, antibodies, disease control, fluorescence microscopy, gelatin, gene expression, genes, immunization, lethal dose 50, muscles, mutants, pH, plasmids, polyacrylamide gel electrophoresis, proteolysis, recombinant vaccines, serine proteinases, turbot, virulence
A putative serine protease gene was cloned from the genomic DNA of Vibrio parahaemolyticus FYZ8621.4. The gene consisted of 1779 base pairs and encoded a 592 amino acid protein. The gene was expressed in Escherichia coli. The expressed protease was purified by Ni-NTA His-Bind Resin column and showed a 63 kDa band on SDS–PAGE. The protease exhibited proteolytic activity on gelatin agar plate and showed maximal proteolytic activity at pH 8.0 and 37 °C. It hydrolyzed N-α-benzoyl-l-tyrosine p-nitroanilide (BAPNA), but did not N-benzoyl-l-arginine ethylester (BAEE), N-benzoyl-l-tyrosine ethylester (BTEE) and N-acetyl-l-tyrosine ethylester (ATEE). Mutants at conserved residues Asp⁵¹ (Asp⁵¹–Asn), His⁸⁹ (His⁸⁹–Asp) and Ser³¹⁸ (Ser³¹⁸–Leu, Ser³¹⁸–Pro) lost proteolytic activities completely. The protein was confirmed to belong to serine protease. The purified serine protease was toxic to zebrafish with a LD₅₀ of 15.4 μg/fish. A DNA vaccine was constructed by inserting the mutated serine protease (Ser³¹⁸–Pro) gene into pEGFP-N1 plasmid. The pEGFP-N1/m-vps was transfected in HeLa cells. The serine protease was confirmed to be expressed by fluorescence microscopy observation and Western blotting analysis. The pEGFP-N1/m-vps was further observed to express in muscle of the injected turbot (Scophthalmus maximus) by Western blotting seven days after immunization. Efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-vps, with the highest relative percent survival (RPS) of 96.11%. Significant specific antibody responses were also observed in the turbot vaccinated with the DNA vaccine. The results indicated that the serine protease might be a potential virulence factor and could be used as an efficient vaccine candidate for the disease control caused by V. parahaemolyticus.