Molecular Mechanism Of Intestinal Mucosal Immune System And Oral Nanoparticles Vaccine Preparation In Pelodiscus Sinensis

Pelodiscus sinensis,commonly known as Chinese soft-shelled turtle,is the most traditional freshwater aquaculture varieties raised in China.However,owing to intensive cultivation and environmental deterioration in recent decades,it frequently suuffered from serious infectious diseases caused by bacteria?viruses?parasites and other pathogens,which resulted in great economic loss and threatened the sustainable development of aquaculture every year.Intestinal mucosal immune system,including humoral immunity and cellular immunity,is the first defense line of body against foreign microbial.The study of intestinal mucosal immune system of P.sinensis can help us understand its immune defense mechanism better and provide the theoretical basis for developmet of oral nano-vaccine.In this study,the methods of histology,molecular biology and transcriptomics were used to study the intestinal mucosal immune system of P.sinensis systematically.The main immune cells in the intestine were figured out and molecular mechanism of intestine immune respons to exogenous stimuli on the intestinal tract after exogenous stimulate was revealed.In addition,the immune response of the important protein polymers immunoglobulin receptor(pIgR)involved in mucosal immunity was analyzed.On the basis of the above,we also developed a nano-oral vaccine carrying the outer membrane protein of pathogenic bacteria,and tested its immune effect in P.sinensis.The results are as follows:1.Detection of intestinal mucosal immune related Cells and geneIn this chapter,we mainly study on the immune cells in the intestinal mucosal immune system.At first,transmission electron microscopy was used to observe the intestinal cross section.And we found that there were a lot of intraepithelial lymphocytes(IEL)and goblet cells(GC)in the intestinal tract.HE staining and PAS staining was used to calculate the number of IEL and GC in the intestine respectively.The results showed that the number of the IEL and GC increased firstly and then decreased,rising to the peak at 48 h,which was significantly higher than that of the saline control group(P<0.05).We also used RT-PCR to detect the expression of immunoglobulin IgM and IgD in the intestinal tract after exogenous stimulate.The expression of IgM gene was increased,which suggest that it might be very improtant in intestinal mucosal immunity.2.Analysis on intestine of P.sinensis after intragastric challenge with with transcriptome sequencingIn the present research,histological analysis and transcriptome analysis was performed on the small intestine of P.sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens.The result showed the number of intraepithelial lymphocytes(IELs)and goblet cells(GCs)in its intestine increased significantly at 48 h post-challenge with LPS by intragastrical route,indicating clearly the intestinal immune response was induced.Compared with the control,a total of 748 differentially expressed genes(DEGs)were identified,including 361 up-regulated genes and 387 down-regulated genes.Based on the Gene Ontology(GO)annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG),48 immune-related DEGs were identified,which were classified into 82 GO terms and 14 pathways.Finally,18 DEGs,which were randomly selected,were confirmed by quantitative real-time PCR(qRT-PCR).Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P.sinensis.3.Cloning of pIgR gene and its immune response to exogenous stimuliThrough the analysis of amino acid structure and homology,we found that the pIgR gene could encode 591 amino acids.There was a signal peptide sequence located in the front section of coding region.The predicted molecular mass of pIgR protein is 65.7 kDa,with an estimated PI of 7.38.pIgR is composed of extracellular domain,transmembrane and intracellular areas,and the extracellular domain includes four domains.pIgR in P.sinensis has higher homology with it in other reptiles,followed by birds,mammals and amphibians,and has the lowest homology with it in fish.With RT-PCR,we found that the expression of pIgR gene in the tissues from high to low was:small intestine,esophagus,large intestine,liver,heart,kidney,muscle,spleen,stomach and kidney.In the process of embryonic development,the expression of pIgR raised firstly and then decrease,and reached the peak at 31 d after hatching.The expression of pIgR mRNA was significantly increased in the esophagus,stomach,small intestine and large intestine after stimulation with Aeromonas sobria and LPS.The results showed that the expression of pIgR protein was expressed in the cytoplasm of intestinal epithelial cells and the expression of pIgR protein in the small intestine increased with the stimulation of LPS with western blotting and immunohstochemistry,which further suggest the important role of the protein in the mucosal immunity of P.sinensis.4.Preparation and identification of immunological effect of Oral nano-vaccine in P.SinensisIn this chapter,an ion exchange method was used to prepare a carboxymethyl chitosan nanoparticles.The optimum ratio is carboxymethyl chitosan 0.5 mg/mL and CaCl2 2 mg/mL.The average particle size of the nanoparticles is 229.07 ± 4.28 nm,the PDI is only 0.070 ± 0.02.The particle size is small,the dispersity is good and the particles are uniform.We have verified that the particles have many good characteristics,such as safety,stability and adhesion to the intestinal tract.The outer membrane protein(OMP)with the main protein size of 43 kDa and 38 kDa from A.hydrophila was extracted,and the nano-oral vaccine by embedding the protein with carboxymethyl chitosan nanoparticles was prepared.Finally,with the saline,carboxymethyl chitosan nanoparticles and outer membrane protein as control,the histochemistry and fluorescence quantitative PCR technology were used to test the immune effect of oral nano-vaccine.The results showed that the immune effect of nano-vaccine was better than the outer membrane Protein and carboxymethyl chitosan,and secondary feeding of nano-vaccines can also cause the intestinal immune response.