Effect of OnCL-K1 on the killing of bacteria in vivo

After a 12-h period of siRNA injection, each group of tilapia was i.p. infected with S. agalactiae in the presence or absence of (r)OnCL-K1. The levels of acid phosphatase and NO were determined in the head kidney and spleen at 12 h postinfection. The head kidney and spleen of each group were lysed and centrifuged, and the resulting supernatant was used for acid phosphatase detection. In addition, the NO in head kidney and spleen was detected using a total NO assay kit (Beyotime). Briefly, the preprepared tissue homogenate (40 μl) was added to diluent (20 μl), NADPH (5 μl, 2 mM), FAD (10 μl), and nitrate reductase (5 μl) in turn, mixed, and incubated at 37°C for 30 min. Then, lactate dehydrogenase buffer (10 μl) and lactate dehydrogenase (10 μl) were added and incubated at 37°C for another 30 min. Finally, Griess reagents I and II were added and incubated at room temperature for 10 min. The reaction was detected at OD₅₆₀ using a microplate reader, and the concentration of NO in the sample was calculated according to the standard curve. Furthermore, the expression levels of EAA1, M6PR, Lamp-1, Dynactin, and v-ATPase in the head kidney of each group were detected by qRT-PCR at 12 h postinfection. Moreover, for the survival assay, fish were randomly divided into three groups (20 fish for each group). O. niloticus was injected i.m. with 100 μl of dsOnCD93 (20 ng), or PBS, respectively. After 12 h, fish were infected with 100 μl of S. agalactiae (1 × 10⁸ CFU/ml) via i.p. injection in the presence of (r)OnCL-K1. The daily deaths of tilapia were recorded.