Expression, Purification, Site-directed Mutagenesis Of Vibrio Anguillarum EmpA Metalloprotease In Escherichia Coli And Development Of Its DNA Vaccine

Vibrio anguillarum is the causative agent of vibrosis, one of the major bacterial disease affecting marine and freshwater fishes in the world. The infected fish showed a rapid fulminating hemorrhagic septicemia and wide-spread tissue damage, and died in several days. The distribution of vibrosis is worldwide, which cause great economic loss to the aquaculture industry. Vibrio anguillarum strain W-1 was originally isolated from diseased seabass (Lateolabrax japonicus). Previous studies revealed that the extracellular protease EmpA of Vibrio anguillarum was one its important virulence factors. The gene coding EmpA was cloned and sequenced.In this study, empA was cloned into three prokaryotic expression plasmids pBV220, pBAD24 and pET24d(+) respectively, resulting three recombinant Escherichia coli strains JM109/pBV220/empA,TOP10/pBAD24/empA and BL21(DE3)/pET24d(+)/empA. We finally chose BL21(DE3)/pET24d(+)/empA to express, purify and mutate empA since comparisons of the expression of empA in this three recombinant strains showed that BL21(DE3)/pET24d(+)/empA had the best expression profile. The expression conditions of the recombinant E. coli BL21(DE3)/pET24d(+)/empA were optimized. It was found that the soluble productions of EmpA expressed in recombinant cells and secreted to the medium were both maximal when E. coli BL21(DE3)/pET24d(+)/empA was induced with 1 mM IPTG at 25℃. The expressed reombinant EmpA could react with the anti-EmpA serum in Western-blot. EmpAs expressed in recombinant E. coli and secreted to the medium were purified by metal chelating affinity chromatography. Estimated molecular weights of purified EmpAs were both 36 kDa by SDS-PAGE, but both 44.6 kDa if the samples were not heated in boiling water. And they were both determined to be 44.6 kDa by Sephadex G-200 gel filtration. Thus, EmpA expressed in E. coli was a monomer polypeptide. Purified EmpA had cytotoxicity to flounder gill cell line (FG). The development of CPE-like morphologic damage of the cells was visible by light microscopy within 12 h of incubation of the cells with 5μg of purified EmpA. It could also cause death of turbot when injected intraperitoneally, which showed similar symptoms with diseased fish infected by Vibrio anguillarum. The LD50 of EmpA to turbot was established as 8.1μg protein/g of fish.The nucleotides of the amino acid residues in the active sites of EmpA were mutated by PCR site-directed mutagenesis. All recombinant plasmids with mutated empAs were transformed into E. coli BL21(DE3), and mutated EmpAs 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 reduced the proteolytic activity and cytotoxicity of the enzyme with different levels, indicating that they play an important role in enzymatic activity.To construct DNA vaccine, mutated empA (m-empA7) was subcloned into eukaryotic expression vector pEGFP-N1, resulting the recombinant plasmid pEGFP-N1/m-empA7. It was then transfected into CHO and HEK293T cells and the expression of m-empA7 was detected with fluorescent microscope, RT-PCR and Western-blot, which indicating that m-empA7 was expressed in CHO and HEK293T cells.Flounder were intramuscularly injected with 50μg of the DNA vaccine solution to examine if m-empA7 could be expressed. Fluorescent microscope, RT-PCR and Western-blot detection revealed that m-empA7 was expressed in the muscle, spleen, kidney, hind intestines, heart, gill and liver of flounder. To study the protective efficacy of the recombinant plasmid pEGFP-N1/m-empA7 on flounder, fish were respectively immunized with three doses of DNA vaccine (5μg/fish, 20μg /fish and 50μg /fish). The results of ELISA revealed that the immunized fish harvested much higher special antibody against m-EmpA7 in relation to negative controls of PBS buffer and pEGFP-N1, and the antibody titer increased as the immunized dose increased. Five weeks after vaccination, fish were challenged by intramuscularly injection of Vibrio anguillarum W-1 cell suspension. Mortalities following exposure to the bacteria were much lower in fish vaccinated with pEGFP-N1/m-empA7 compared to those of the control groups injected with PBS buffer and pEGFP-N1 alone. DNA vaccine of three doses protected fish from the bacterium with 57.1%, 71.4% and 85.7% of RPS respectively.