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| Proteins, Expression, Isolation and Analysis | To determine the presence of any soluble MjsvCL protein in the circulating plasma, an immunoprecipitation assay was performed with the MjsvCL Ab to concentrate the target protein to a detectable level. The hemolymph from WSSV-infected shrimp (0, 6, 12, 24, 48, and 72 h) was collected and centrifuged at 800 3 g for 5 min. The supernatant was centrifuged again at 20,000 3 g for 20 min to obtain the plasma. Ten shrimp were used for each sample. The plasma was diluted with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40, 1 mM EDTA, 0.5 mM PMSF, pH 8.0) and precleared with 50 ml protein A beads (GenScript, Nanjing, China) with agitation for 40 min at 4?C. After removing the beads by centrifugation at 12,000 3 g for 10 min, 5 ml MjsvCL antiserum was added to the supernatant. The mixture was incubated overnight with agitation at 4?C. Protein A beads (10 ml) were then added to isolate the Ab with agitation at 4?C for 1 h. The beads were collected, washed four times using RIPA buffer with each wash lasting 10 min, resuspended in SDSPAGE sample buffer (15 ml), and boiled for 10 min before Western blot analysis using the MjsvCL Ab. | Get A Quote |
White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus–associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to v... More
