Screening Of The Main Protective Antigens Of Aeromonas Veronii TH0426 And Immunogenicity Analysis Of A Recombinant Lactobacillus Casei Expressing OmpF Of Strain TH0426

Aeromonas veronii is a common ubiquitous pathogen of humans,animals,and fish including other aquaculture,causing systemic infections in aquatic environments,determining its antigenic proteins are significant for recombinant vaccine development to reduce economic losses in fish.Due to problems of antibiotic resistance,and the rapidity with which the infection spreads among fishes,vaccination remains the most effective strategy to combat this infection in fish populations.Among numerous virulence factors associated with bacterial virulence,outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity.In this study,we identified immunogenic proteins of Aeromonas veronii strain TH0426 by immunoproteomic approach.Initially whole cells vaccine of Aeromonas veronii strain TH0426 was prepared advantuated with Freund’s adjuvant,and immuned Cyprinus carpio by intraperitoneal injection.Fish were immunized twice(1-14 day)throughout the experiment by 0.2 m L intraperitonially.Weekly blood samples were collected to obtain convelesent sera.The ELISA results reveal that Cyprinus carpio sera found significantly higher after first immunization;it peaked after booster dose and remained stable in the serum.I-D immunoblot analysis shows Cyprinus carpio convelesent sera strongly reacted with abundant of the immunogenic proteins.Furthermore,2-D immunoblot analysis shows that the entirety 39 immunoreactive spots of the protein were identified by two-dimensional(2-D)electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-TOF-MS).Proteins MW were found between 102.83 to 25.09 k Da.The next part of the study was focused on the construction of the recombinant Lactobacillus L.casei/p PG612-Omp F vaccine.Initially,Omp F gene of A.veronii from the identified proteins was amplified cloned and sequence analyzed.Using recombinant DNA technology,we successfully constructed the prokaryotic expression vector,which could simultaneously express Omp F gene of Aeromonas veronii TH0426.In addition,Escherichia coli-Lactobacillus shuttle expression vector p PG612 system based on the glucose operon promoter expressing Omp F gene.The recombinant Lactobacillus L.casei/p PG612-Omp F based on Lactobacillus casei from Cyprinus carpio gut as expressed vector and Omp F protein was verified by SDS-PAGE and Western blot.The results demonstrated that transformed Omp F gene was efficiently expressing its protein of ~45KDa size possessing strong reactogenicity.We evaluated the potential protective immunity response of recombinant L.casei L.casei/p PG612-Omp F.To identify whether the recombinant strains have the ability to induce immune responses,Carassius auratus were challenged with A.veronii TH0426 after orally immunized with recombinant L.casei L.casei/p PG612-Omp F.The first immunization was carried out on the first three consecutive days followed by two sets of booster doses on 18 th,19th,20 th and 35 th,36th and 37 th.To detect the level of specific antibodies and the expressions of immune-related genes in serum and tissues,blood and organs samples were collected on 1 d,7 d,12 d,17 d,21 d,31 d and 38 d days after immunization.To evaluate colonization potential of recombinant Lactobacillus casei in Carassius auratus intestines,intestinal lavage was collected before immunization and 1 d,3 d,5 d,7 d,12 d,16 d and 20 d days after immunization.The results showed that recombinant L.casei colonized effectively in foregut as compared with mid and hindgut respectively.The presence of L.casei/p PG612-Omp F was found peaked in 7th day on foregut,whereas control group(PBS)gastrointestinal tract was not associated with any other gut content or tissue type.Subsequently,we assessed immune responses;specific Ig M,Ig T,IL-8 and IFN-γ activity in serum were determined by ELISA and cytokines ELISA.The results suggested that the fish immunized with recombinant L.casei could produce high level Ig M after booster and second immunization.Moreover,the level of Ig T,IL-8 and IFN-γ were up-regulated after booster immunization.Simultaneously,the expression levels of some immune relevant genes(IL-1β,IL-10,TNF-α,IFNγ)in heart,liver,spleen,head kidney and gut tissues were assessed by quantitative real-time PCR.The results revealed that relative m RNA expression of IL-1β was enhanced in heart,liver and head kidney after immunization.The expression of IL-10 in heart,liver,spleen,head kidney and gut tissues L.casei-p PG612-Omp F group of fish was observed up-regulation(P <0.01)on 31 day.After 21 days,the expression of TNF-α gene was seen up-regulated in heart,spleen,head kidney and gut tissues.The relative m RNA expression of IFNγ was found significantly upregulated in heart,liver,spleen and head kidney after 12 days.Furthermore,immunized fish were challenged with a lethal dose of infectious A.veronii TH0426 1.0×106 cells at a dose of 5X LD50 0.2 ml of PBS / fish.The cumulative mortality and relative percentage survival was 51.43 % in L.casei/p PG612-Omp F group whereas death rate reached 100% in other groups.In this study,recombinant Lactobacillus L.casei/p PG612-OmpF,expressing Omp F gene,has been constructed.The recombinant L.casei could induce specific Ig M and humoral immune response,resulting in partly protecting Carassius auratus from A.veronii.These results revealed that mucosal immunization with new recombinant live L.casei-p PG612-Omp F is an effective oral vaccine candidate against Aeromonas veronii.