Application Of Recombinant Lactobacillus Casei In The OmpW Gene Of Aeromonas Veronii

Aeromonas veronii is a symbiotic pathogen of human-plant-fish that has been discovered in recent years and can cause the onset of human and various aquatic organisms.The bacteria are widely found in natural waters,soils and aquatic animals.As an adhesin of Aeromonas veronii,outer membrane protein W is a potential protective antigen and can be tested as a genetic engineering vaccine candidate antigen.Lactobacillus is considered to be a probiotic that can tolerate the body's defense mechanisms.By oral means,they can enter the body and survive in large numbers.They are also closely linked to the intestinal mucosa,which regulate intestinal flora balance and enhance immunity and other probiotic functions.Therefore,it is of great significance to develop a vector for Lactobacillus plantarum as an antigen presented by the Aeromonas veronii.In this study,the recombinant Lactobacillus casei in the OmpW gene of Aeromonas veronii conserved in the Veterinary Research Laboratory was used to verify the secretion of Lc-pPG612-OmpW and cell surface expression Lc-MCS-OmpW.The expression of OmpW gene in Lactobacillus casei was verified by PCR,double digestion and Western blotting.The results showed that the approximately 600 bp OmpW gene fragment was amplified by PCR,and the approximately 22 kDa proteins expressed by the two recombinant Lactobacillus casei strains all had good reactogenicity.In order to test whether the constructed recombinant bacteria could induce mucosal and systemic immune responses,this experiment used carp as a test animal for immunological tests.The Lc-pPG612-OmpW and Lc-MCS-OmpW recombinant strains were induced by lactose and mixed with sodium alginate-coated pellet feed.The tissues and blood were collected 0 d before immunization,and 7 d,16 d,25 d,34 d after immunization,detection of changes in related tissues and serum immunological parameters.Detection of recombinant Lactobacillus casei colonization in the intestine of salmon,setting experimental groups individually,feeding continuously for 7 d,28 d after fasting,the foregut,midgut and hindgut were aseptically collected and counted using resistant plates.The challenge test measured the immune protection rate.Serum IgM antibody levels,ACP,AKP,SOD,LZM,IL-10,IL-1?,TNF-?,and IFN-? were measured using ELISA.The results showed that after oral immunization,all the indicators were significantly improved.Immunization of squid by oral administration can induce IgM and various immune parameters in humoral immunity of squid.At the same time,the ability of phagocytosis and differentiation of leukocytes was significantly enhanced.The level of transcription of IL-10,IL-1?,TNF-? and IFN-? cytokines in each tissue was detected by qRT-PCR.The results showed that the recombinant Lactobacillus casei stimulated the mucous membrane by colonization in the intestine,which increased the transcriptional level of cytokines,thus enhancing the immunity of the carp.The colonization situation also showed that after 28 d of fasting,recombinant lactobacillus was still present in the intestine,indicating its good colonization ability in the body;After 20 d of challenge,recombinant Lactobacillus casei can effectively alleviate the acute infection caused by Aeromonas veronii,and have a protective effect to some extent a protective effect.In summary,this study validated the recombinant Lactobacillus casei in the OmpW gene of Aeromonas veronii Lc-pPG612-OmpW and Lc-MCS-OmpW preserved in the laboratory.Oral immunization of eel can induce humoral immune response,produce high levels of IgM and upregulate the activity of immune-related enzymes.Meanwhile,this method promotes leukocyte differentiation,enhances its phagocytic activity,and improves the body's humoral immune response,and the transcription level of cytokines in various tissues also shows a trend of upward adjustment.Recombinant lactobacillus casei can effectively protect carp and improve its immunity.The results lays a foundation for the development and application of aquaculture vector vaccines and oral lactic acid bacteria vaccines.