| This paper reports the immunosuppressant screening and immune regulation by lectin inhibitor in the White Shrimp Litopenaeus vannamei, discussed mainly the following theses: (1) Establishment of haemocyte immune activity cell culture in vitro of Litopenaeus vannamei; (2) phenoloxidase inhibitor and lectin inhibitor screening of haemocyte from white shrimp Litopenaeus vannamei; (3) The effect of combination of LPS and lectin inhibitors on prophenoloxidase activating system of haemocytes in Litopenaeus vannamei.1. Establishment of haemocyte immune activity keeping methods in vitro of Litopenaeus vannameiThe effects of four media on haemocyte counts, viability and exocytosis of prophenoloxidase cascade system were examined. It was founded that all four media have notable effects on haemocyte counts, viability, and exocytosis of prophenoloxidase (proPO) cascade system (P<0.05). During 0-6 h, the haemocyte counts, viability and prophenoloxidase activity in four treatments declined gradually, while phenoloxidase activity climbed increasingly. However, the haemocyte counts and viability in second medium were significantly higher than those three media, during 3-6h, haemocyte counts of each medium were stabilized, then decreased gradually after 6h; in 0-3h, the hemagglutinating activity of the first,third and forth medium increased, the second one did not change, then kept stability after decreased at 6h, during 0-6h, prophenoloxidase activity of the first medium was higher than another ones, while the phenoloxidase activity was the lowest, at 12h prophenoloxidase activity of the second medium was highest, and there was no significant difference between the second and the other three groups. So, it's suggested that the second medium can be chosen as a cell culture solution of Litopenaeus vannamei haemocyte.2. The research of phenoloxidase and lectin inhibitor screening of Litopenaeus vannamei haemocyteEffect of phenoloxidase and lectin inhibitor on haemocyte counts, viability and prophenoloxidase cascade system were studied. The three concentrations of phenoloxidase inhibitors p-APMSF had a significant influence on both haemocyte counts and viability of the Litopenaeus Vannamei. As the incubation time went on, the counts and viability of each management group decreased gradually. The hemagglutinating activity of the 50μm and 100μm groups remained the same, while the 25μm group was the least among the treated groups. The prophenoloxidase activity and serine proteinase activity of the 25μm group were much higher than the others and had significant negative correlation with p-APMSF'concentration. The three kinds of lectin inhibitors had a significant influence on haemocyte counts and viability of Litopenaeus Vannamei(P<0.05). The counts and viability in each treated group decrease gradually in the experimental time. Each treated group's phenoloxidase activity increased gradually while the prophenoloxidase activity of haemocyte decreased gradually. The glucose treated group was completely inhibited to the hemagglutinating activity of Litopenaeus Vannamei. Therefore, we choose glucose to do experiment to the shrimp lectin inhibitors.3. Effect of combination of LPS and lectin inhibitor on the Prophenoloxidase cascade system of Litopenaeus VannameiThe combination of LPS and lectin inhibitor had a great influence on prophenoloxidase cascade system of Litopenaeus Vannamei(P<0.05). In the experiment period, haemocyte counts and viability of each management group decreased gradually. Compared with the LPS treated group, the counts and viability of the combination of LPS and lectin inhibitor was lower. Compared with the control group, each treated group's hemagglutinating activity decreased. In 3 hours, the hemagglutinating activity of LPS treated group and the control group increased, while the hemagglutinating activity of the combination of LPS and lectin inhibitor decreased. During 3 to 6 h, the hemagglutinating activity of each group did not change much. LPS and combination of LPS and lectin inhibitor had a great influence on phenoloxidase activity, prophenoloxidase activity and serine protease activity of haemocytes (p<0.05). In the experiment period, both the proPO activity and Serine protease activity of each treated group decreased, and compared with the control group, LPS treated group decreased lower while all of the PO activity increased. In each treated group, phenoloxidase activity shows a positive correlation with the incubation time, while the serine protease activity and proPO activity were negatively correlated with the incubation time. Therefore, lectin plays an important role in the immune of Litopenaeus Vannamei. |
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