Construction And Immune Efficacy Of Recombinant Lactobacillus Casei Expressing Aha1 Of Aeromonas Veronii With CTB/LTB As Molecular Adjuvant

Aeromonas veronii is a pathogen that can infect human,animal and aquatic organisms.It poses a huge threat to the health of many aquatic organisms such as Cyprinus carpio,and can also cause a variety of diseases in humans and animals,such as necrotizing fasciitis,dysentery and sepsis.Aha1 is one of the main components of the outer membrane protein,which has strong immunogenicity and has good prospects in vaccine development.However,oral vaccines require a large number of antigens to stimulate the body to produce a higher level of immune response,and mucosal immune adjuvants can stimulate the systemic and local mucosal immune response and improve the immune effect.Cholera toxin B subunit and Escherichia coli heat labile enterotoxin B subunit,as a common mucosal immune adjuvant,have good development prospects.In this study,selected the aha1 gene of A.veronii TH0426 strain as the target gene and fused with the gene of CTB or LTB,respectively.At the same time,used Lactobacillus casei as the antigen presentation carrier to construct recombinant Lactobacillus casei LC-pPG-Aha1,LC-pPG-Aha1-CTB and LC-pPG-Aha1-LTB of surface-displayed expressing and their protein expression was tested by Western blot and immunofluorescence methods.Our results showed that the nucleotide sequence of the gene of Aha1 or the fusion gene of Aha1-CTB and Aha1-LTB was 1038 bp,1449 bp and 1383 bp,respectively.And the protein weight of the recombinant Lactobacillus casei were 44 kDa,57 kDa,and 59 kDa,respectively.The target protein could be anchored on the surface of bacteria by immunofluorescence methods.The genetic stability results showed that the recombinant Lactobacillus casei had good stability.To evaluate the immune effects of the recombinant strains,we will immunize Cyprinus carpio via orogastric route with the recombinant strains which was induced by lactose and coated with pellet feed.Each immunization was 3 times,and the immunization cycles interval was 14 days,and the immunization was conducted 3 days.The serum and different tissue samples in Cyprinus carpio were collected before 0 days immunization and after 7,14,21,28,35 and 42 days immunization,and detected specific IgM levels in serum and ACP,AKP,SOD,LYS,C3,C4 and Lectin enzyme activities by ELISA,and detected IL-10,IL-1?,TNF-?,IgZ1 and IgZ2 cytokine levels in various tissues via qRT-PCR.The intestinal tissues of Cyprinus carpio were collected after 1,3,7,12 and 18 days immunization and intestinal value detection was performed.To detect the protective effect after the end of immunization 5 days,the challenge test was performed.Our results demonstrated that recombinant strains could increase the level of IgM antibodies;non-specific immune indicators in the serum showed an upward-regulated trend,indicating that recombinant strains can increase the humoral immunity level of Cyprinus carpio.The expression of immune-related cytokines in different tissue increased to varying degrees,indicating that recombinant strains can enhance the cellular immunity of Cyprinus carpio.The colony count of each intestine segment showed that recombinant strains could colonize the intestine of Cyprinus carpio,and the number of recombinant strains in the front,middle and hind intestines was inconsistent,and the midgut and hindgut had a strong ability to set values.After the challenge,it was observed that the recombinant strains could resist the infection against Aeromonas veronii,and the relative protection rates were 53.57 %,64.29 %,and 60.71 %,respectively.Various immune indicators of LC-pPG-Aha1-CTB and LC-pPG-Aha1-LTB were higher than LC-pPG-Aha1,indicating that the addition of mucosal immune adjuvants can enhance humoral and cellular immunity.To sum up,we have constructed successfully the recombinant L.casei(LC-pPG-Aha1,LC-pPG-Aha1-CTB and LC-pPG-Aha1-LTB),which can induce Cyprinus carpio to produce higher levels of IgM antibodies after oral immunization.And it also enhances the activity of AKP and other related enzymes,promotes the transcription level of related cytokines in fish,and enhances immunity.The research results provide a theoretical basis for the development of oral lactic acid bacteria delivery vector vaccines and the application of mucosal immune adjuvants in the aquaculture industry.