The trypsin-like serine protease domain of Paralichthys olivaceus complement factor I regulates complement activation and inhibits bacterial growth

Complement C3 and Activated Fragment C3a Are Involved in Complement Activation and Anti-Bacterial Immunity

“…In lamprey, C3 knockdown decreased the survival rate of larvae against the infection with A. hydrophila (77). In pervious study, we found that recombinant trypsin-like serine protease domain of Fcator I inhibited complement activation by degrading C3b in the serum of Japanese flounder (45). Based on the previous results, in this study, we found that in Japanese flounder administered with pPoC3si, the mRNA level of PoC3 was significantly decreased, suggesting that PoC3 expression was successfully interfered by the small RNA expressed from the plasmid.…”

“…Purification of PoC3 and preparation of mouse anti-PoC3 polyclonal antibody were described previously (45,46). V. harveyi, E. tarda, E. coli, S. iniae, V. anguillarum and P. fluorescens were grown to mid-log phase as described above.…”

“…After incubation, the bacteria were centrifuged and washed with PBS for 3 times. The collected bacteria were lysed with 8 M urea, centrifuged at 10,000 rpm for 2 min, and the supernatant was taken for Western blot analysis (45). The interaction between serum PoC3 and bacteria was detected by ELISA as reported previously (47).…”

“…Serum was prepared from Japanese flounder containing pPoC3si, pPoC3siC or PBS (control) and was diluted serially in Hank's Balanced Salt Solution (HBSS) (Solarbio, Beijing, China). Hemolytic activity and bactericidal activity assay were determined as reported previously (45,48). For hemolytic activity, the control sample at 1/4 serum dilution was defined as 100% for easy of comparison.…”

“…In order to examine the binding effect of PoC3 to bacteria in the serum, the different bacteria were incubated with the serum of Japanese flounder or PBS (control). The western blot analyses with mouse anti-PoC3 polyclonal antibody (45) showed that PoC3 in the serum can bind to V. harveyi, E. tarda, E. coli, S. iniae, V. anguillarum and P. fluorescens, among which V. anguillarum had the strongest binding ability, but no binding phenomenon was found in the control group (Figure 4A). In addition, PoC3 could be degraded into activated fragments after incubation with bacteria in serum, including C3c and C3d.…”