Prokaryotic expression of outer membrane protein Tol C of Pseudomonas plecoglossicida and its immunoprotective effect analysis on Larimichthys crocea

In recent years, due to the high density of mariculture, the water circulation is not smooth, the pollution of the sea area is becoming more and more serious, and the disease occurs frequently. Visceral white spot disease caused by Pseudomonas plecoglossicida is a common bacterial disease of Larimichthys crocea in cage culture. In order to evaluate the immunoprotective effect of Tol C (GenBank: EPB97112.1), a recombinant Tol C domain protein named B6 was obtained by prokaryotic expression. The immunogenicity of B6 was preliminarily detected by rabbit anti-P. plecoglossicida polyclonal antiserum. Followed, a subunit vaccine was prepared by mixing B6 with alum adjuvant, and L. crocea was immunized by intraperitoneal injection. The relative protection rate (RPS) was detected 4 weeks after immunization. Meanwhile, the serum, head kidney and spleen of fish were collected at 2, 4, 6 and 8 weeks after immunization, and the serum antibody titer as well as the expression level of immune-related genes were determined. Finally, the head kidney and spleen of healthy individuals and challenged ones were both collected and made into paraffin sections to compare histopathological changes. The results showed that the molecular weight of B6 was 22 ku. Western blot results showed that the rabbit antiserum against P. plecoglossicida could strongly bind to B6 with a single special band. 4 weeks after inoculation with B6 subunit vaccine, the RPS reached 72.22%. ELISA showed that the serum antibody titer increased significantly since the 2nd week after immunization, peaked at the 4th week (log₂ 8.65), and then decreased slightly to log₂ 6.98 at the 8th week. RT-PCR showed that the expression level of immune-related genes in head kidney and spleen were both up-regulated with varying degrees at 2, 4, 6 and 8 weeks after immunization. Among them, the up-regulation rates of IL-1β and MHC I α was the largest, with 93.33 and 77.02 times each in head kidney, and 241.13 and 131.95 times each in spleen, respectively. Except for CD8, the expression up-regulation trend of IgM, IL-1β, CD4, MHC I α and MHC II β in spleen was all higher than that in head kidney. Paraffin sections showed that severe bleeding spots and nodules appeared in the head kidney and spleen of the control group after challenge, accompanied by different degrees of siderophore deposition. Comparatively, there were only a few bleeding spots in the head kidney and spleen of the B6 immunized group, and no nodules and siderophore deposition. In conclusion, our study first reported the prokaryotic expression and immune protection evaluation of the outer membrane protein Tol C from P. plecoglossicida, the results showed the recombinant Tol C protein had strong immunogenicity and good immune protection in L. crocea, with a RPS of 72.22 % 4 weeks after immunization, thus is a potential candidate of subunit vaccine.