Figure 1.
SDS–PAGE analysis of the purified Sp-SPH protein.
The recombinant Sp-SPH protein was purified by using Ni2+ affinity chromatography via a 6× His tag as described above. Lane M, molecular weight marker; lane 1, purified Sp-SPH protein; lane 2, cultured medium from pPIC9K/Sp-SPH recombinant clone induced by methonal before protein purification.
Figure 2.
Binding activity of recombinant Sp-SPH protein to different bacteria.
The recombinant Sp-SPH protein was incubated with formaldehyde-fixed bacteria. After incubation, the supernatants were separated by centrifugation. The pellets were then washed with PBS and the bound proteins were eluted with SDS-PAGE loading buffer followed by electrophoresis. (A) All eluted samples were examined by Western blot analysis under reducing condition with the employment of the anti-His antibody. –s: bacterium + Sp-SPH protein; -p: bacterium + PBS. (B) Summery of binding affinity of Sp-SPH protein to the bacteria selected.
Figure 3.
Analysis of the binding affinity between recombinant Sp-SPH protein and the bacterial or fungal associated molecules.
The binding affinity of Sp-SPH protein to LPS, PGN or β-1, 3-glucan was tested by ELISA. Absorbance of each well was measured at 450 nm with a Multifunctional microplate reader (GENios). The binding parameters, apparent dissociation constant Kd, and the maximum binding (Amax), were determined by non-linearly fitting as A = Amax [L]/(Kd+ [L]). Diamond:lipopolysaccharide (LPS); Square: peptidoglycan (PGN); Triangle: β-1, 3-glucan. The data were representative of the average value of four repeated experiments. Bars indicated mean ± S.E. (n = 4).
Figure 4.
Determination of hemocyte adhesion activity of the mud crab mediated by Sp-SPH recombinant protein.
Different concentrations of Sp-SPH recombinant protein were used for coating the ELISA plate followed by addition of crab hemocyte suspension. After washing, the cell adhesion was assessed by measuring the OD595nm value with Multifunctional microplate reader (GENios). *: Significant differences in hemocyte adhesion of Sp-SPH protein treated samples compared to that of non-Sp-SPH protein control (paired t-test, P<0.05). This experiment was repeated for four times. The results were shown as means ± standards errors (n = 4).
Figure 5.
Phenoloxidase activity enhanced by Sp-SPH protein in the HLS of mud crab.
PGN, Sp-SPH, PGN/Sp-SPH, PGN/Sp-ALF2, or PGN/BSA was added to the mud crab HLS, respectively. The PO activity of HLS was then determined by using L-dopa as substrate and defined as U/mL. Significant difference in PO activity was marked as stars (paired t -test, P<0.05). HLS: hemocyte lysate supernatant; PGN: peptidoglycan+HLS; SPH: Sp-SPH+HLS; PGN+SPH: peptidoglycan+ Sp-SPH protein+HLS; PGN+ALF2: peptidoglycan+antilipopolysaccharide factor2+HLS; PGN+BSA:peptidoglycan+bovine serum albumin+HLS. This experiment was repeated three times and the data represented means of triplicates. Bars indicated mean ± S.E. (n = 3).
Figure 6.
Cumulative mortality of mud crab challenged with bacteria pre-coated with Sp-SPH protein.
Crabs were challenged with A. hydrophila, V. parahemolyticus or V. alginolyiicus pre-coated with Sp-SPH protein as described above. Ten animals were used for each group. The mortality was recorded hourly. The cumulative mortality of crabs, in which the bacteria were coated with Sp-SPH protein (square, SPH), was compared with that of animals treated with “PBS-coated” bacteria (diamond, PBS). A: The cumulative mortality caused by A. hydrophila; B: The cumulative mortality caused by V. parahemolyticus; C: The cumulative mortality caused by V. alginolyiicus. This experiment was repeated three times and the data represented means of triplicates. Bars indicated mean ± S.E. (n = 3).