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Various species of the Vibrio genus cause serious infectious diseases that affect both sea animals and humans. In particular, Vibrio splendidus is a pathogenic bacteria that can trigger many diseases in a variety of sea animals, This bacterial species can cause infected fish to exhibit a number of pathologies, including bleeding tails, rotting and even ulcerated skin, congestion of the base of the dorsal fin, pectoral fin and tail fin, swelling and erosion of the liver, erosion of the kidneys, and others. Hemolysin, which is produced by a number of pathogenic bacteria, is one of the primary pathogenic agents that cause these health issues. However, there are very few reports about the hemolysin activity of V. splendidus. There has been a report involving construction of a V. splendidus mutant lacking a metalloproteinase gene with a counterselectable suicide vector. To our knowledge, this is the first full report about 4-hydroxyphenylpyruvate dioxygenase （4HPPD）, which is the gene of V. splendidus hemolysin. The results as follows:1 A bacterial strain named QD-1 was isolated from the diseased Sciaenops ocellatus with ulceration in Qingdao. Strain QD-1 was identified as Vibrio splendidu with morphological, physiological and biochemical methods, whith named Vibrio splendidus QD-1.2 pathogenicity test by pathogenic bacterium:Hemolysis activity assay: We performed hemolytic examination of bacteria on sheep blood plates according to the existing literature. To prepare each plate, we prepared 100 ml of liquid medium at 121°C for 20 min, cooled the mixture to 50°C, added 5-10 ml of defibrinated sheep blood, shook the mixture, and finally poured the plate. The bacteria were spread flat over the plate, and were incubated at 28°C overnight. The colonies that exhibited a ring around them were considered positive for hemolysis, the ambient bacterial colony had a transparent cycle, and its radius was 2mm. Phospholipase activity assay: We performed an assay to measure the activity of phospholipase according to previous studies. Briefly, we prepared 2216E agar-solidified medium, added 1% of fresh egg yolk, and made the surface flat. The colonies that exhibited ivory translucent circles around them were considered positive for phospholipase activity. the radius of the translucent cycle was 0.5 mm.Pathogenicity test: For the pathogenicity test, fish were divided into six groups, including five experimental groups and one control group. Each group included 10 healthy guppies, and each group was maintained in different aquariums. The bacterial preparation was diluted to 2×104, 2×105, 2×106, 2×107, and 2×108 cfu/mL, and the dilutions were injected into the experimental guppies （200μL for each）, whereas the fish in the control group were injected with normal saline solution. When a guppy died, bacteria were isolated from the fish. Of note, the water temperature was kept at 27.4-28°C. We observed and recorded the morbidity and mortality daily, and subsequently calculated the LD50 according to the simplified Korbor’s formula method. Based on statistical analysis of the number of the deaths in the fish, we calculated the LD50 using Korbor’s approach, and determined that the LD50 was 5.62×105 cfu/mL.3 The V. splendidus strain was inoculated into a seawater medium AGAR plate, and was kept at 28°C for 24 h. We picked a single colony and inoculated it into 5 ml of seawater medium, subsequently optimized the conditions according to a previous study, and kept the preparation at 28°C for 18 h. A 1% inoculate was prepared in 1L seawater medium, and was maintained with 200 r/m shaking for 48 h.Ammonium sulfate precipitation: The culture was centrifuged at 8,000 rpm for 30 min, the supernatant was collected, and solid ammonium sulfate was added into the supernatant at 70% saturation. Furthermore, the precipitate was solubilized at a concentration of 50 mmol/L in Tris-HCL in （pH 7.8） buffer fluid, and was kept in the same buffer for dialysis at 4°C for 48 h. The enrichment of this preparation wassubsequently used for coarse extraction of beta-exotoxin. Sephadex-G100 chromatography: Coarse extraction exotoxin was obtained using a Sephadex-G100 chromatography column with an enrichment culture concentration of 50 mmol/L in Tris-HCL buffer （pH=7.8）. Elution was carried out at a velocity of 6 drops/min, and each was collected in a 2-ml tube, its OD value was determined in its peak place, with PEG100 enrichment merger protein concentration, namely because purification glue is toxic. The purity of the resultant exotoxin was detected by SDS-PAGE. This revealed an obvious protein band at 40.7 kD. We found that the OD value of purified hemolysin protein was 0.8 mg/mL.The purified hemolysin was diluted by 10, 100, and 1000 times for aseptic filtration, and was divided into four groups. Each dilution was separately injected into five guppies using intraperitoneal injection. Specifically, each fish was injected with 100μL of a dilution, and was observed for 48 h at room temperature. We recorded the number of experimental fish that died in each group, and calculated the LD50 for group according to Korbor’s formula method. At the same time, the diluent only was injected into five guppies for negative control. To determine the exotoxin LD50, we performed statistical analysis of the number of fish deaths, and calculated the LD50 of the guppies on the basis of Korbor’s formula method. We determined that the LD50 was 6.35μg.4 We also obtained results of the original nuclear expression vector pGEX-6P-H in hemolysin. Through exploration of a variety of conditions, we determined to use 0.8 mmol/L IPTG, and to oscillate it at 200 rpm at 30°C for 4 h, which were the optimal conditions we found. Increasing the IPTG concentration caused no increase in the expression quantity. Furthermore, similar expression results were obtained from oscillating the preparation 4 h and overnight.When the pure beta-exotoxin antigen was diluted 50 times （16μg/mL） and the antibody was diluted 160 times, the measurement P/N was the largest （the positive serum OD value was 0.591, whereas the negative control value was 0.058）. Furthermore, indirect ELISA revealed that the titer of the anti-hemolysin antigen of the immune mice was 1:3 200. Upon vaccinating the guppies with the mixtures, we found that all the fish in the filtration group died, the group given the 10 times diluted antigen had two deaths, and the group given the 100 times diluted antigen had one death. In contrast, all the negative-control fish survived.
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