Effect Of C-type Lectin On The Immune Response Mechanism Of White Shrimp Litopenaeus Vannamei

This paper reports the mechanism of immune response mechanism modulatedby C-type lectin in the White Shrimp Litopenaeus vannamei, discussed mainly thefollowing theses:(1) Prokaryotic expression of C-type lectin gene of Litopenaeusvannamei;(2) prophenoloxidase (proPO) activating system and their signaltransduction in white shrimp Litopenaeus vannamei haemocytes induced bylipopolysaccharide and recombinant C-type lectin protein;(3) prophenoloxidase(proPO) activating system and signal transduction of Litopenaeus vannameiinduced by lipopolysaccharide and recombinant C-type lectin protein.1. Prokaryotic Expression of C-type lectin gene of Litopenaeus vannamei andimmunologic activity evaluation of Recombinant ProteinLitopenaeus vannamei C-type lectin gene (C-type lectin) was expressed inEscherichia coli and the immunologic activity of the recombinant protein wasanalyzed. Results showed that SDS-PAGE revealed the molecular weight of LvLecprotein is39kD, which is consistent with the predicted size. Western blot revealed itcould bind with anti-His antibody, which proved that C-type lectin recombinantprotein in Escherichia coli succeeded in expressing. For the hemagglutination assay,no obvious agglutination was detected on examined erythrocytes. No obviousagglutination could be observed when Vibrio alginolyticus, Vibrio harueyi, Vibrioanguillarum, Staphylococcus aureus were incubated with LvLec protein.2. Effect of the recombinant C-type lectin on the haemocyte immune responseof white shrimp Litopenaeus vannameiWe based on cell suspension culture to study the effect of C-type lectin on thehaemocytes immune response of Litopenaeus vannamei. The experiment was dividedinto four groups, control group, recombinant protein (LvLec protein,1.0mg/mL), Lipopolysaccharide (LPS,1.0mg/L), LPS and Lvlec protein(LPS+Lvlec protein，1.0mg/L+1.0mg/mL). After0,3,6,9,12,24hours, we harvested the solutions and thenassessed parameters respectively. The results showed that Lvlec protein had asignificant effect on haemocyte count, cell viability, phagocytic activity and thehamagglutinating activity of white shrimp Litopenaeus vannamei(P<0.05). Theextracelluar phenoloxidase activity of the Lvlec protein group showed peak changesduring3-12h, at9h the extracelluar phenoloxidase activity was highest, thenrecovered to the control level(P<0.05). The concentration of cGMP and PKA in threetreatment groups showed peak changes during3-12h, they reached the maximumvalue at9h. In conclusion, the present study documented that LPS and Lvlec protein had apositive correlation with the immune response, they could activated prophenoloxidase,and probably played an important role in immune response.3. Effect of recombinant C-type lectin injection on the immune responsemechanism of white shrimp Litopenaeus vannameiIn order to study effect of injection of C-type lectin on the immune responsemechanism of Litopenaeus vannamei, the experiment was divided into four groups,control group, recombinant protein (LvLec protein,1.0mg/mL), Vibrio harueyi (V.harveyi,10~6cfu/mL), Vibrio harueyi and Lvlec protein (V. harveyi+Lvlec protein，10~6cfu/mL+1.0mg/mL).The results showed that V. harveyi and Lvlec proteininjection had significant effect on the hemocyte count, prophenoloxidase activity,phenoloxidase activity. V. harveyi and Lvlec protein injection resulted in a significanteffect on hamagglutinating activity, phagocytic activity, bacteriolytic activity and theconcentration of CGMP, the activity of PKA (P<0.05). The results showed that Lvlecprotein correlated with decreases in haemocyte count, changes in PO activity,hamagglutinating activity, phagocytic activity and the concentrations of cGMP andPKA. We analysed that C-type lectin had a positive correlation with the immuneresponse. The results showed that Lvlec protein correlated with decreases inhaemocyte count, changes in PO activity, hamagglutinating activity, phagocytic activity and the concentrations of cGMP and PKA. We speculated that the pathway ofC-type to the immune response may be Lvlecâ†'cGMPâ†'PKAâ†'POâ†'thehamagglutinating activity and phagocytic activity.