Expression, Purification, Site-directed Mutagenesis Of Serine Protease And Hemolysin Of Vibrio Parahaemolyticus And Development Of Bivalent Vaccines

Vibrios are Gram-negative halophilic bacteria and common causative agents of vibriosis, which cause great economic loss to the aquaculture industry. Vibrio parahaemolyticus is an organism which causes potentially serious infections in aquiculture animals and humans.In this study, a putative serine protease gene (vps) was cloned from the genomic DNA of a pathogenic V. parahaemolyticus FYZ8621.4. The gene consisted of 1779 base pairs and encoded a 592 amino acid protein. The gene was inserted into prokaryotic expression plasmid pET-28a(+) and expressed in Escherichia coli BL21(DE3). The expressed serine protease was purified by Ni-NTA metal affinity chromatography 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℃. The purified serine protease also showed hemolytic activity on blood plates. The protein was confirmed to belong to serine protease. The purified serine protease was toxic to zebra fish with a LD50 of 15.4μg fish-1. The nucleotides of the amino acid residues in the catalytic triad of serine protease were mutated by PCR site-directed mutagenesis. All mutants (Asp⁵¹ -Asn, His⁸⁹-Asp, Ser³¹⁸-Ala, Ser 318-Leu and Ser 318-Pro) were transformed into E. coli BL21(DE3), and mutated serine protease were expressed and purified to characterize the effects of alterations of these amino acid residues on enzymatic activity. It was found that all of the single point mutations at the conservative residues lost or reduced the proteolytic activity and cytotoxicity of the enzyme, indicating that they played an important role in enzymatic activity. These evidences demonstrated that the putative protease was one of serine proteases in V. parahaemolyticus. A DNA vaccine was constructed by inserting the mutated serine protease (Ser³¹⁸-Pro) gene into eukaryotic expression vector pEGFP-N1. The pEGFP-N1/ m-vps was transfected in HeLa cells. The serine protease was confirmed to be expressed by fluorescence microscopy observation. The protective efficiencies of such vaccine against V. parahaemolyticus challenge were evaluated in turbot (Scophthalmus maximus). Fish immunized with a single intramuscular injection of different doses (10 and 50μg/fish) of the DNA vaccine showed significant serum antibody levels after vaccination, compared to fish vaccinated with the control vector pEGFP-N1. The pEGFP-N1/m-vps was further observed strong expression in muscle of the injected turbot by reverse transcription PCR (PT-PCR) and Western-blot seven days after immunization. Four weeks post-inoculation, the efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-vps. The relative percent survivals (RPSs) of two different dose groups were 80.55 % and 96.11 % (P<0.05), respectively. Significant specific antibody response was also observed in the turbot vaccinated with the DNA vaccine. The highest titers of antibody with different doses were 1: 512 and 1: 1,024, respectively. The results indicated that the serine protease might be a potential virulence factor and the mutated ptotein could be used as an efficient vaccine candidate for the disease caused by V. parahaemolyticus.A thermostable direct hemolysin gene (tdh2) was cloned from the genomic DNA of V. parahaemolyticus FYZ8621.4. The gene consisted of 570 base pairs and encoded a 189 amino acid protein. The gene was inserted into the vector pET-28a(+) and expressed in E. coli BL21(DE3). The expressed protease was purified by Ni-NTA metal affinity chromatography and showed a 21 kDa band on SDS-PAGE. Mutants at conserved residues Gly⁶² (Gly⁶² -Ser) and Trp⁶⁵ (Trp⁶⁵-Ala) lost hemolytic activities on blood plates completely by PCR site-directed mutagenesis. A DNA vaccine was constructed by inserting the mutated serine protease (Trp⁶⁵-Ala) gene into the vector pEGFP-N1. The protective efficiencies of such vaccine against V. parahaemolyticus challenge were evaluated in turbot. Fish immunized with a single intramuscular injection of different doses (10 and 50μg/fish) of the DNA vaccine showed significant serum antibody levels after vaccination, compared to fish vaccinated with the control vector pEGFP-N1. The pEGFP-N1/m-thd2 was further observed strong expression in muscle of the injected turbot by PT-PCR and Western-blot seven days after immunization. Four weeks post-inoculation, the efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-tdh2. The RPSs of two different dose groups were 80.55 % and 96.11 % (P<0.05), respectively. Significant specific antibody response was also observed in the turbot vaccinated with the DNA vaccine. The highest titers of antibody with different doses were 1: 512 and 1: 1,024, respectively.TDH gene (tdh2) and serine protease gene (vps) of V. parahaemolyticus FYZ8621.4 were inserted into the vector pET-28a(+), successively, then expressed in E. coli BL21(DE3). The expressed protease was purified by Ni-NTA metal affinity chromatography and showed an 85 kDa band on SDS-PAGE, which corresponded to a deduced molecular weight of the fusion protein of TDH2 and serine protease. The fusion gene tdh2-vps was inserted into the vector pEGFP-N1 to construct a bivalent DNA vaccine. The protective efficiencies of the bivalent DNA vaccine against V. parahaemolyticus challenge were evaluated in turbot. Fish immunized with a single intramuscular injection of different doses (10 and 50μg/fish) of the tdh2-vps DNA vaccine showed significant serum antibody levels after vaccination, compared to fish vaccinated with the control vector pEGFP-N1. The pEGFP-N1/thd2-vps was further observed strong expression in muscle of the injected turbot by PT-PCR and Western-blot seven days after immunization. Four weeks post-inoculation, turbot vaccinated with the bivalent DNA vaccine were challenged with pathogenic V. parahaemolyticus by intraperitoneal injection. The RPSs of two different dose groups were 92.22 % and 100 % (P<0.05), respectively. Significant specific antibody response was also observed in the turbot vaccinated with the DNA vaccine. The highest titers of antibody with different doses were 1: 4,096 and 1: 8,192, respectively.TDH gene tdh2 of V. parahaemolyticus FYZ8621.4 and oligopeptide permease (Opp) gene oppA of V. harveyi SF-1 were inserted into the vector pET-28a(+), successively, then expressed in E. coli BL21(DE3). The expressed protease was purified by Ni-NTA metal affinity chromatography and showed an 84 kDa band on SDS-PAGE, which corresponded to a deduced molecular weight of the fusion protein of TDH2 and OppA. The fusion gene tdh2-oppA was inserted into the vector pEGFP-N1 to construct a bivalent DNA vaccine. The protective efficiencies of such vaccine against V. parahaemolyticus and V. harveyi challenges were evaluated in turbot. Fish immunized with a single intramuscular injection of different doses (10 and 50μg/fish) of the tdh2-oppA DNA vaccine showed significant serum antibody levels after vaccination, compared to fish vaccinated with the control vector pEGFP-N1. The pEGFP-N1/thd2-oppA was further observed strong expression in muscle of the injected turbot by PT-PCR and Western-blot seven days after immunization. Four weeks post-inoculation, turbot vaccinated with the bivalent DNA vaccine were challenged with pathogenic V. parahaemolyticus and V. harveyi, respectively. The highest RPS rate of 100 % against V. parahaemolyticus and 60 % against V. harveyi was recorded (P<0.05). Significant specific antibody response was also observed in the turbot vaccinated with the DNA vaccine. The highest titers of antibody with different doses were 1: 512 and 1: 2,048, respectively.TDH gene (tdh2) of V. parahaemolyticus FYZ8621.4 and outer membrane protein (OMP) gene (ompU) of V. anguillarum W-1 were inserted into vector pET-28a(+), successively, then expressed in E. coli BL21(DE3). The expressed protease was purified by Ni-NTA metal affinity chromatography and showed a 57 kDa band on SDS-PAGE, which corresponded to a deduced molecular weight of the fusion protein of TDH2 and OmpU. Then the fusion gene tdh2-ompU was inserted into the vector pEGFP-N1 to construct a bivalent DNA vaccine. The protective efficiencies of such vaccine against V. parahaemolyticus and V. anguillarum challenges were evaluated in turbot. Fish immunized with a single intramuscular injection of different doses (10 and 50μg/fish) of the tdh2-ompU DNA vaccine showed significant serum antibody levels after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pEGFP-N1. The pEGFP-N1/thd2-ompU was further observed strong expression in muscle of the injected turbot by PT-PCR and Western-blot seven days after immunization. Four weeks post-inoculation, turbot vaccinated with the bivalent DNA vaccine were challenged with V. parahaemolyticus by intraperitoneal injection and V. anguillarum by intramuscular injection, respectively. The highest RPS rate of 100 % against V. parahaemolyticus and 35 % against V. anguillarum were recorded (P<0.05). Significant specific antibody response was also observed in the turbot vaccinated with the DNA vaccine. The highest titers of antibody with different doses were 1: 1,024 and 1: 2,048, respectively.