Construction And Immune Efficacy Of Recombinant Lactobacillus Casei Experssing OmpA? Of Aeromonas Veronii C5-?/? As Molecular Adjuvant

Aeromonas veronii(A.veronii),a pathogen that can infect both human and fish,has brought huge losses to aquaculture industry and posed a huge threat to public health.Because of the limitation of fish vaccine conditions,the development of oral vaccine is of great significance.Outer membrane protein A is a kind of adhesion protein,which can enhance the bactericidal efficiency and has good immunogenicity.It has potential as an antigen gene of DNA vaccine.However,because of the low expression of DNA vaccine protein,it can not stimulate the body to produce a strong enough immune response.Molecular adjuvants can enhance the antigenic effect by activating the innate immune system,including interferon,complement molecules and interleukin,so it has good prospects.In this study,the OmpA? gene of A.veronii TH0426 and complement C5-?with C5-? gene of carp was cloned.OmpA? was overlapped and amplified with C5-? and C5-? genes respectively by SOE-PCR.Our results demonstrated that the nucleotide sequence of fusion gene was 1500 base pairs,which expressed about 58 kDa protein has possessed well reaction-ogenicity.The indirect immunofluorescence test also showed that the expressed protein was displayed on the cell surface.To identify whether the recombinant strains have the ability to induce immune responses,carassius aumtus was inoculated with the recombinant strains via oral route.At 0 d before immunization and 7 d,16 d,25 d and 34 d after immunization,the tissue and blood were collected to determine immune-related indexes.To assess immune responses,specific IgM,ACP,SOD and LZM activity in serum were determined by ELISA.The results suggested that the carassius aumtus immunized with recombinant Lactobacillus casei could produce high level IgM in the serum.And the activity of ACP,SOD and lysozyme were up-regulated.The results indicated that recombinant Lactobacillus could raise humoral immunity;The phagocytic activity of leukocytes were significantly enhanced compared with the control group;The expression levels of some immunerelevant genes were assessed by quantitative real-time PCR.The results indicated that the transcription level of IL-10,IL-1?,TNF-? and IFN-? were up-regulated.The recombinant Lactobacillus increased the cytokine transcription level and enhanced the cellular immune response.To detect the colonization of recombinant L.casei,the experimental group fasted for days,prosogaster,mid-gut and hind-gut were collected for colony counting.The results showed the colonization of recombinant L.casei was remakable in instine and the colonization ability was good.Subsequently,the immunized carassius aumtus were inoculated intraperitoneally with A.veronii TH0426,and all the immune groups were protected to some extent.The addition of molecular adjuvants C5-? and C5-? increased the immune index compared with the recombinant L.casei expressing OmpA? gene.The result indicated that complement C5 as a molecular adjuvant can effectively improve the immune effect of recombinant L.casei.Activates innate immunity while effectively promoting cellular immune responses.In summary,the recombinant L.casei Lc-pPG-1-OmpAI-C5-?/? and Lc-pPG-2-OmpAI-C5-?/? with OmpA? gene C5-? and C5-? as molecular adjuvants can keep IgM at a high level,and increase the level of serum enzyme activity and cytokines after oral immunization.Moreover,molecular adjuvants can stimulate a strong immune response and improve the immune effect of the vaccine.This study provides a theoretical basis for the development and utilization of oral Lactobacillus vaccine,and the application of molecular adjuvants.