Construction Of A.veronii Malt Gene Recombinant Lactic Acid Bacteria With IFN-γ As Molecular Adjuvant And Evaluation Of Immune Effect

Aeromonas veronii is a pathogenic bacterium that is commensurate with humans,animals,and fish.It can exist in the water body for a long time and infect aquatic organisms such as fish,shrimp,crabs and turtles,which seriously endangers the development of aquaculture.Although the rational use of antibiotics can reduce the loss caused by some pathogenic A.veronii,people use a lot of antibiotics to cause the bacteria to develop resistance.At the same time,antibiotics cannot solve the problem of rapid spread of bacteria after fish infection.Aquatic vaccines,as a quick and effective way to prevent bacterial infections in fish,can solve these problems without harming the fish itself.Maltoporin is a kind of outer membrane pore adhesion protein of A.veronii,which has strong immunogenicity antigen.Molecular adjuvants can increase the protein expression of genetically engineered vaccines,and enhance the stability and safety of vaccines.Therefore,the research of molecular adjuvants has become a research hotspot in enhancing vaccine immunity.In this study,we cloned the malt gene and the crucian IFN-γ gene of A.veronii TH0426 strain.The malt gene and IFN-γ gene were overlapped and amplified by SOE-PCR.The results showed that the target fragment of about 1800 bp was successfully amplified.After prokaryotic expression of the malt gene,L.casei 393 with three recombinant plasmids p PG-malt,p PG-INF-γ and p PG-malt-INF-γ was constructed.The sequencing results showed that the recombinant L.casei was successfully constructed.In order to study the effect of molecular adjuvants to enhance the vaccine,the recombinant lactic acid bacteria were induced by lactose and coated on fish feed.The carassius aumtus was immunized by oral immunization.The immunized carassius aumtus was immunized at 0 d before immunization and 7 d.Serum and tissue collection of liver,spleen,kidney,and gut.The content of Ig M,ACP,AKP,SOD,LYS,C3,C4 in serum was determined by ELISA method.The results showed that recombinant L.casei Lc-p PG-malt and Lc-p PG-malt-IFN-γ could immunize the Ig M antibody in the serum of carassius aumtus.The level remains high,and the activities of AKP,ACP,SOD,LYS,C3,and C4 are significantly up-regulated,indicating that the recombinant L.casei Lc-p PG-malt and Lc-p PG-malt-IFN-γ can enhance the humoral immunity of carassius aumtus.At the same time,the phagocytic activity of leukocytes was significantly stronger than that of the control group.The expression of immune genes in each tissue of the immunized carassius aumtus was measured by q RT-PCR,and the results showed that the each tissue of the transcription levels of IL-8,IL-10,IL-1β,TNF-α,TRAF-6 and My D88 showed a significant upward trend,indicating that recombinant L.casei can increase the transcription level of cytokines and enhance cellular immune response.Plate colonies were counted in the hindgut,and the results showed that the recombinant L.casei could colonize well in the intestine.Then the immunized carassius aumtus was inoculated with A.veronii TH0426 in the abdominal cavity.It has a protective effect to a certain extent,and the relative immune protection rate of Lc-p PG-malt-IFN-γ group is higher.The addition of IFN-γ,a molecular adjuvant,improves its immune indexes compared with recombinant L.casei Lc-p PG-malt,indicating that IFN-γ as a molecular adjuvant can effectively improve the immune effect of recombinant L.casei.It can effectively promote cellular immune response while activating innate immunity.However,the results of the Lc-p PG-IFN-γ group and the Lc-p PG group were almost the same,and there was no immune enhancement effect.In summary,this study constructed A.veronii Malt gene recombinant L.casei and malt gene recombinant L.casei with IFN-γ as a molecular adjuvant,which can be used as oral vaccines to maintain a high level of Ig M in fish,and at the same time,it can also make serum enzyme activity and cytokine levels are on the rise,enhancing the differentiation and phagocytosis of white blood cells,and the addition of molecular adjuvants can stimulate the body to produce a stronger immune response and enhance the immune effect of the vaccine.The innovation of this study is: firstly,IFN-γ gene and A.veronii malt gene was fused into L.casei,which was used as an aquatic vaccine to immunize carassius aumtus.The research results are designed to evaluate the immune ability of the new genetic engineering vaccine and provide a theoretical basis for the application of L.casei live vector vaccine in aquaculture.