| Larimichthys crocea is an important mariculture species in China,with its production ranked first for past years in seawater fish species,thus shows prominent economic value.In recent years,with the increasing scale and density of aquaculture,various diseases have erupted frequently.Among them,the mortality rate of visceral white spot disease caused by Pseudomonas plecoglossicida is as high as 70%-80%,which is the most serious bacterial disease in large yellow croakers,causing serious economic losses to the industry.At present,drugs such as enrofloxacin and doxycycline hydrochloride are generally used in production to prevent and control the disease.However,long-term use of drugs will not only increase the risk of drug residues,but also pollute the water ecological environment and may induce new drug-resistant strains.Therefore,it is urgent to develop a safe and efficient precaution method.As an important means of disease prevention and control,the vaccine has been widely used in aquaculture.Subunit vaccine has the advantages of strong specificity and high safety,which is one of the important directions of vaccine development.In a previous study,we obtained the recombinant outer membrane proteins Tol C and Porin of P.plecoglossicida by prokaryotic expression,and verified by Western Blot that the two proteins had strong binding bands with rabbit antiserum to P.plecoglossicida,which suggested that the two recombinant proteins had good immunogenicity.In this study,we further evaluate the immune protection effect of the two recombinant proteins on large yellow croaker.First,starting from the prokaryotic expression strains of Tol C and Porin,sufficient recombinant Tol C and Porin proteins were obtained by optimizing the expression and purification conditions.They were emulsified with alum adjuvant to prepare subunit vaccines.In order to determine the appropriate challenge does under the experimental water temperature conditions,the large yellow croaker was pre-challenged with different gradient concentrations of P.plecoglossicida.According to the pre-challenge data,the appropriate soaking concentration was selected to challenge the large yellow croaker after 4 weeks of immunization,and the relative percent survival(RPS)of 14 days was counted.At the same time,the serum,head kidney and spleen of fish at 2,4,6 and 8 weeks after immunization were collected to determine the serum antibody titer,bactericidal activity,natural immune enzyme activity and the expression of immune-related genes in head kidney and spleen.The head kidney and spleen of the immunized group and the control group after challenge were collected and made into paraffin sections to observe the histopathological changes.The results showed that after 4 weeks of immunization,the water temperature was 16 ±3℃,the RPS of the Tol C vaccine group was 72.22%,and the RPS of the Porin vaccine group was 38.89%.ELISA results showed that the serum antibody titer of the Tol C vaccine group increased significantly from the second week after immunization,peaked at the fourth week(log2 8.65),and then decreased slightly to log2 6.98 at the eighth week.The antibody titer of Porin vaccine group reached the peak at the 4th week,and the antibody titer reached log27.99.The results of serum bactericidal activity showed that the survival rate of P.plecoglossicida in the Tol C immunized group was 31.24%.The lowest survival rate of P.plecoglossicida in the Porin immunized group was 55.32%.The results of natural immune enzyme activity showed that the activities of AKP and ACP in the Tol C immunized group and Porin immunized group were up-regulated and significantly higher than those in PBS control group(P<0.05).The results of RT-PCR showed that the expression of immunerelated genes in the head kidney and spleen was up-regulated to varying degrees at 2,4,6 and 8 weeks after immunization in the Tol C immunization group.Among them,the upregulation of IL-1β and MHCⅠ α was the largest,reaching 93.33 times and 77.02 times in the head kidney,241.13 times and 131.95 times in the spleen,respectively.Except for CD8,the expression of IgM,IL-1β,CD4,MHC I a and MHC Ⅱβin spleen was higher than that in head kidney.The up-regulation of IgM,IL-1β and MHC Ⅰα in the spleen of the Porin vaccine group reached 48.35 times,47.43 times and 113.96 times,respectively.Paraffin sections showed that the PBS control group showed severe bleeding points and vacuolization in the spleen after challenge,cell disorder,and accompanied by a large area of siderophore deposition.The Tol C immunized group had only a small amount of bleeding points in the head kidney and spleen.The Porin immunization group can be observed in the spleen’s serious bleeding,and vacuolization,compared with the PBS control group cells arranged neatly.In summary,this study evaluated the immune protection effects of the outer membrane proteins Tol C and Porin of P.plecoglossicida in large yellow croaker.The results showed that both Tol C and Porin could activate the innate and acquired immunity of the fish,which was manifested as activating natural immune enzymes,inducing serum antibody production,stimulating the expression of immune factors in the head kidney and spleen,and reducing the attack damage of the head kidney and spleen.Among them,the protective effect of Tol C was better than that of Porin,and the 14 d RPS reached 72.22%.After immunization,the serum antibody titer and bactericidal activity increased significantly,which had the potential as a candidate vaccine. |
