Design And Construction Of Vibrio Mimicus Targeted Epitopes Gene Vaccine And Evaluation On Its Immune Efficacy

Vibrio mimicus?V.mimicus?is an enteric pathogen which seriously endangering the development of aquaculture.Intestinal mucosal immunity induced by gene vaccines through oral or anus injection plays a vital role in the resistence to the pathogen.Therefore,the development of a safe and efficient V.mimicus gene vaccine is of great significance in the prevention and control of the pathogen infection in fish.Gene vaccine is well-known for some advantages,such as safety,easy construction,comprehensive and lasting immune response.However,naked gene vaccine through oral or anus injection is susceptible to degradation by digestive enzymes,resulting in decreased immunogenicity.The key measure to solve this problem is to use the suitable carrier which can target the gene vaccine to the antigen presenting cell?APC?for high efficiency expression and presentation.It has been proved that Bacterial Ghosts?BGs?have the function of preventing the degradation of gene vaccine and targeting its expression in APC,while the Invariant chain like protein?Iclp?in fish has the effect of targeting the antigen peptide to MHC molecules and improving the efficiency of antigen presentation.Thus,the Iclp and BGs can be used as internal and external targeting vectors of gene vaccines to enhance their immunogenicity.In previous studies,we have identified the immunodominant epitopes of two immune protective proteins?OmpU adhesion protein and VMH hemolysin protein?of V.mimicus.On this basis,the vaccine target was designed with the dominant epitopes of OmpU protein and VMH protein,and the targeted epitope gene vaccines of V.mimicus were constructed using Iclp and BGs as the internal and external target vectors.Then,the immune effect of the targeted epitope gene vaccines was evaluated.The primary research work and results completed are summarized as follows:1)Design of a tandemly arranged multi-epitope peptide of V.mimicus.Here,three kinds of linkers were used to combine three dominant linear B-cell epitopes and two mimotopes of the VMH protein,as well as two dominant linear B-cell epitopes of the OmpU protein with different tandem ways.Then the feasibility of various combinations was analyzed by using the DNAstar Protean program,The results showed that the epitopes in the tandem of^173-192OmpU-AAY-^90-98OmpU-AAY-^125-137VMH-AAY-^238-244VMH-AAY-^459-470VMH-GGGG S-^mimotope1VMH-GPG-^mimotope2VMH?named OVepis?were relatively independent,and the antigenic parameters were relatively good,which could be used as vaccine targets.2)Construction of three targeted epitopes gene vaccine of V.mimicus.In this paper,the recombinant eukaryotic expression plasmid pcDNA-Ovepis was firstly constructed by enzyme digestion and enzyme connection method,hereafter termed naked epitopes gene vaccine.Then,OVepis gene was inserted into Iclp gene of grass carps and replaced partly gene fragment?from 261 to 280 bp?of CLIP region to form a 1017 bp chimeric gene?named Iclp1-261-Ovepis-Iclp280-711?.The recombinant plasmid pcDNA3.1-Iclp-Ovepis was constructed,hereafter termed endogenous-targeted DNA vaccine.Finally,Vaccine plasmids pcDNA3.1-Ovepis and pcDNA3.1-Iclp-Ovepis were respectively loaded into lyophilized DH5?-BGs to form DH5?-BGs/pcDNA3.1-Ovepis?hereafter termed exogenous-targeted epitopes gene vaccine?and DH5?-BGs/pcDNA3.1-Iclp-Ovepis?hereafter termed double-targeted epitopes gene vaccine?by the membrane capsule method.The results of double enzyme digestion and sequencing showed that there was no mutation and deletion in both OVepis and Iclp-OVepis genes.The fluorescence quantitative PCR detection showed that each milligram BGs could load 214?g pcDNA3.1-OVepis and 166?g pcDNA3.1-Iclp-OVepis.The results showed that the three Vibrio mimicus targeted epitopes gene vaccines were successfully constructed.3)Evaluation on the immune effect of targeted epitopes gene vaccines.In this study,three targeted epitopes gene vaccines and naked epitope vaccine were used to immunize grass carp by anus injection,respectively.The immune effect of each vaccine was evaluated by detecting the transcriptional expression level of immune related genes in different tissues,serum and intestinal mucus non-specific immune factor activity,serum and intestinal mucus antibody titer,Proliferation activity of peripheral blood lymphocytes and intestinal intraepithelial lymphocytes,and the relative immune protection rate of immune grass carp.The results showed that the immunological indexes of grass carp in the double-targeted epitopes gene vaccine group were superior to that of 2 single targeted epitopes gene vaccine groups,and the immune indexes of the 2 single targeted epitopes gene vaccine groups were superior to the naked epitopes gene vaccine group,and the three targeted DNA vaccines and the naked DNA vaccine were used to immune grass carp.The relative immune protection rates were 80%?double-targeted epitopes gene vaccine?,73.3%?exogenous-targeted epitopes gene vaccine?,67.7%?endogenous-targeted epitopes gene vaccine?and 40%?naked epitopes gene vaccine?.In conclusion,three targeted epitopes gene vaccines of V.mimicus were successfully constructed.The immunoprotective potency from strong to weak was double-targeted epitopes gene vaccine,followed by exogenous-targeted epitopes gene vaccine,endogenous-targeted epitopes gene vaccine and naked epitopes gene vaccine.