Vaccine Construction And Immune Effect Analysis Based On Pilin Gene Of Vibrio Harveyi

The pearl gentian grouper is a hybrid breed that has been bred by the male giant grouper(Epinephelus lanceolatus,♂)and the female blotchy rock cod(Epinephelus fuscoguttatus,♀).It is an important mariculture fish in China.With the expansion of its breeding scale,Vibrio diseases have become one of the diseases that seriously affect the healthy development of the industry in recently years.Pilin is one of the most important virulence factors in Vibrio,which plays an important role in the process of bacterial infection.In this study,candidate subunit vaccines and DNA vaccines were constructed based on the pilin of Vibrio harveyi,and the immune effects of these two types of vaccines on the pearl gentian grouper were evaluated.The results were as follows:1.Sequence analysis showed that the open reading frame of pilin is 459 bp,which encodes 152 amino acids.Its molecular weight is 15.78 k Da and the theoretical isoelectric point is 7.94.According to the homology analysis,the pilin of V.harveyi has high homology with other Vibrios,and the highest homology is with V.navarrensis(93.38%).2.Based on pilin,the subunit vaccine rpilin was constructed in this study.After immunizing the fish,the challenge experiment of V.harveyi was conducted on the experimental fish in week 4 and week 8,and the results showed that the average relative immune protection rate reached 78.00% and 59.10%.Immune indexes results showed that after immunization,the activities of acid phosphatase(ACP),alkaline phosphatase(AKP),superoxide dismutase(SOD),and lysozyme(LZM)in the serum of the immune group were significantly up-regulated compared with that of the control group(PBS)(P<0.05,P<0.01).Respiratory outbreak activity in the rpilin group was significantly higher than that in the control group(P<0.05,P<0.01),indicating that rpilin can effectively cause the body’ activity of macrophages.Elisa experimental results show that antibody can be detected in 3～8 weeks after rpilin immuned pearl gentian grouper.Real-time fluorescence quantitative PCR was used to detect the expressions of immune-related genes in the spleen of fish immunized for 4 weeks after challenge,the results showed that TNFα,IL1β,MHCⅠα,and Ig M expression quantity increased significantly compared with the control group.3.In this study,pilin DNA vaccine,p CNpilin was constructed based on pilin.After its immunization,the challenge test of V.harveyi was conducted on the experimental fish in week 8,and the results showed that the average relative immune protection rate was as high as 80.81%.Immune indexes results showed that after immunization,the activities of acid phosphatase(ACP),alkaline phosphatase(AKP),lysozyme(LZM),and superoxide dismutase(SOD)in the serum of the immune group were significantly increased compared with those of the control group(P<0.05,P<0.01).Respiratory outbreak activity in the p CNpilin group was significantly higher than that in the control group(P<0.01).Elisa results showed that antibody can be detected in 3～8 weeks after p CNpilin vaccine immuned pearl gentian grouper.Real-time fluorescence quantitative PCR was used to detect the expression of immune-related genes in the spleen of fish immunized for 4 weeks after challenge.The results showed that the expression levels of TNFα,IL1β,MHCα,and Ig M were significantly up-regulated compared with the control group.In conclusion,our experiment showed that both the subunit vaccine and DNA vaccine prepared with the V.harveyi pilin as the immunogens could improve the immune capacity of pearl gentian grouper,and had a good relative immune protection rate.The research results provided a new idea for the immune prevention and control of fish.