| Vibrio harveyi is a gram-negative, motile rod bacterium in marine and estuarine environments, which has been recognized as one of the important bacterial pathogens of marine animals, including Penaeus japonicus, Holothuria scabra and various species of fish. The pathogen was commonly reported and prevalent in many countries around the world such as China, Thailand, India and Australia, which caused severe losses to aquiculture industries.Some antibiotics played an active role in controlling infections of Vibrio harveyi. Misusage has resulted in drug residue and drug-resistance, which limitsits its application. Current vaccine strategies are oriented to replace conventional vaccines with more effective and safer approaches, such as DNA vaccine, live vector vaccine, gene engineering subunit vaccine and others.Reactive oxygen species (ROS) are generated from endogenous cellular metabolism, and increased under various environmental stress situations, including extreme temperatures, UV-radiation, high light intensities, higher osmotic and toxins. As a consequence of disruption balance between generation and depletion of ROS, Oxidative stress occurs and leads to cell damages. Superoxide dismutases, which abate or clear the effect of superoxide radicals via catalyse the dismutation into hydrogen peroxide and oxygen, belong to the anti-oxidation defense system and considered to be the first line of cellular defense against the oxygen toxicity of reactive oxygen species. SODs exist widely in both prokaryotic and eukaryotic organisms. There are three types of superoxide dismutases, which classed depending on their metal cofactors: copper-zinc-containing SOD(Cu-Zn-SOD),manganese containing SOD(Mn-SOD) and iron-containing SOD(Fe-SOD).Sod gene was cloned by PCR amplification from the chromosomal DNA of Vibrio harveyi. The nucleotide sequence was determined. The ORF of the sod gene consists of 600bp, encoding a polypeptide of 199 amino acids. Sequence analysis showed that homologies of the amino acid sequence with those of Fe-SOD of Vibrio harveyi, V. alginolyticus, V. parahaemolyticus, V. orientalis, V. metschnikovii, V. cholerae,V. mimicus, V. fischeri,V. splendidus ranged from 91% to 99% .The sod gene was subcloned into pET26b(+).The prokaryotic expression plasmid pET26b(+) /sod was transformed into E.coli DH5α, then toBL21(DE3).After a 6-h induction with 1 mmol/L IPTG at 37 degrees, the expressed protein possessed an N-terminal His-tag sequence was purified by Ni(+)-affinity chromatography and identified with SDS-PAGE. The single protein brand was detected by SDS-PAGE, and molecular weight of the purified protein was 27kDa.Pyorgallol autoxidation was used to determine SOD activity of the purified protein. The purified SOD had maximal activity at pH 7.0 and was stable over a range of pH 6-8. The optimal temperature for enzyme activity was 20°C. The protein was stable below 40°C and retained only 3 % activity after one hour at 60°C. Inhibitors and absorption spectroscopy were used for the identification of the metal types in purified SOD: The purified SOD belonged to Fe-SOD based on the analysis of sensitivity to hydrogen peroxid H2O2, chlorofrom-ethanol and absorption peak at 280 nm and 362 nm though ultraviolet-visible spectroscopy.Turbots (Scophthalmus maximus) were immunized with 50 ug of the purified SOD by intramuscular injection . The immunized fish were then challenged with 0.1 mL of V. harveyi (3.9×108 CFU/mL) four weeks later. Challenge result demonstrated that the mortality of the immunized group was 20.00 % and that of control team was 100.00 %, which suggest purified SOD as an antigen offer immune protection to turbots. Specific antibody could be detected in the sera of the immunized fish with Western blot analysis, indicating antibody response was generated. |
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