Differential Proteomics Analysis Of Intestinal Tissue Of Grass Carp Before And After Immunization With Vaccine And Preparation Of Key Protein-Specific Antibodies

Vibrio mimicus is an intestinal pathogen causing vibriosis in aquaculture fish.In our previous study,a double-targeted nucleic acid vaccine of V mimicus by using E.coli DH5a bacterial ghosts and invariant chain-like protein of fish as exogenous and endogenous targeting delivery carriers,respectively.And we confirmed the vaccine could elicit significantly higher intestinal mucosal immune response and immunoprotection than did naked nucleic acid vaccine.However,the molecular regulation mechanism of intestinal mucosal immune enhancement is not clear.The immune effect of oral vaccine mainly depends on the level of intestinal mucosal immunity.Studies have shown that a large number of immune cells and immune molecules are distributed in the intestinal mucosa of bony fishes,which plays a role in intestinal mucosal immunity.However,due to the variety of fish species,the complex and varied physiological characteristics,and the lack of universal marker detection antibodies,the research progress of intestinal mucosal immune response mechanism and regulation mechanism in fish is relatively slow.In recent years,with the rapid development of proteomics technology,especially the introduction of iTRAQ technology,new ideas and methods have been provided for the study on the intestinal mucosal immunity in fish.Based on the preparation of double-targeting nucleic acid vaccine of Vibrio mimicus,grass carp as test object,the comparative proteomics of grass carp intestinal tissue before and after vaccination with the double-targeted nucleic acid vaccine was studied by mass spectrometry-based iTRAQ technology.The key differentially expressed proteins(DEPs)related to immunity and their related immune pathways were excavated,and specific antibodies against key DEPs were developed.The main research work and achievements are summarized as follows:1)Preparation of double-targeted nucleic acid vaccine of V.mimicus.In this paper,according to the method established by Cao Ji in our laboratory,we first prepared E.coli DH5a Bacterial Ghost,and then the target nucleic acid vaccine(pcDNA3.1-Iclp-OVepis)was extracted from Vibrio mimicus.Finally,pcDNA3.1-Iclp-OVepis was loaded into the decidua of DH5a Bacterial Ghost by membrane capsule method to obtain double-targeted nucleic acid vaccine of Vibrio mimicus.2)Immunization,sampling and extraction of total protein from intestinal tissues of grass carp.The grass carp were immunized twice with Vibrio mimimetica double-targeted nucleic acid vaccine by oral administration.On the 28th day after immunization,9 fish were randomly selected from immunization group and PBS control group(as pre-immunization control),and the middle and late intestines were collected.The 3 fish-intestinal tubes were mixed into one sample,and each group had 3 samples in total.The total protein of intestinal tissues of each group was extracted by acetone precipitation method,and the protein extraction effect was detected by SDS-PAGE and BCA protein quantification kit.The results showed that the total protein concentration in intestinal tissues was between 12-15 mg/mL and 6100-6600 ug,and the protein bands were clear and complete,which could be used for subsequent proteomics research.3)Differential proteomic analysis of intestinal tissues in the grass carps before and after immunization.In this paper,mass spectrometry-based iTRAQ technology was used to identify and quantify the proteins in the intestinal tissues of grass carp before and after immunization.The differentially expressed proteins(DEPs)were screened,and MRM validation and bioinformatics analysis were carried out.Results 11949 peptides and 4249 proteins were identified and quantitatively,including 1173 DEPs,629 DEPs up-regulated,544 DEPs down-regulated and 140 immune-related DEPs.The correlation coefficient between the MRM verification results of 7 randomly selected DEPs and the iTRAQ quantitative results is 0.4752,indicating that the correlation between the two is good,and the iTRAQ data is credible.Bioinformatics analysis showed that 1173 DEPs involved 15 cell components,11 molecular functions and 26 biological processes;117 DEPs enriched 300 KEGG Pathway pathways,including 20 immune-related pathways;the interaction network of immune-related DEPs consisted of 61 nodes and 227 edges;Among them,12 proteins such as Hsp90,transforming growth factor β-activated kinase 1(MAP3K7/TAK1)and major histocompatibility complex Ⅱ(MHCⅡ)are the central nodes with high association.4)Preparation of specific antibodies against key DEPs.For the preparation of enrichment in antigen presented respectively and NOD-like receptor signal pathways in the two key immune related DEPs(gcTAK1 protein and gcMHCII-beta protein)of specific antibodies,in this paper,using genetic engineering technology preparation of recombinant protein gcTAKl and gcMHCII-β protein,and immune the New Zealand white rabbit to prepare corresponding polyclonal antibodies.Results a total of 50 mL of rabbit anti-rgctakl antibody and 40 mL of rabbit anti-MHCⅡ-β antibody with ELISA titer of 1:3276800 and titer of 1:204800 were obtained.In summary,based on the differential proteomics study on the intestine tissues of grass carp before and after oral immunization,140 immune-related DEPs were explored,which were mainly enriched in immune-related pathways such as antigen presentation,complement and coagulation cascade,NOD-like receptor signaling pathway and MAPK signaling pathway.In addition,specific antibodies against two key immune-related DEPs were prepared.These results laid a good foundation for further study on the molecular regulation mechanism of intestinal mucosal immune enhancement induced by the double-targeted nucleic acid vaccine.