Study On The Mucosal Adaptive Immunity Mediated By Immunoglobulint In Common Carp(Cyprinus Carpio L.)

Fish are the most ancient vertebrate that have immunoglobulins,adaptive immunity is very important to protect fish from pathogens.Mucosal associated immunity in fish is more developed for their aquatic living conditions.Great amount of immunoglobulins distribute in mucosal associated lymphoid tisssues of teleost fish.These antibodies fromboth systemic immune organs and mucosal tissuesare specific antibodies to protect the fish from pathogens.Compared to the mammals,the discovery of fish mucosal specific antibody is later,its immune function and the differencesbetween IgT and IgM are still unknown.On the other hand,there are two seperate lymphocyte subgroups in fish depending on the marker of membrane immunoglobulin,IgM positive lymphocytes and IgT positive lymphocytes.It is necessary to have more further studies of the two antibody secreting cells and it will help us understand the evolution of vetebrate immune system.Common carp(Cyprinus carpio L.)is one of the main aquaculture fish in China,Aeromonas hydrophila is also a common pathogenin aquaculture.Prophylactic immunization method have great value for fish in prevention from infectious diseases.The research on carp immune responses mediated by antibody will helpus to make correct dicisionfor combating disease.IgT(IgZ),discovered recently,is regarded as a specific antibody function in mucosal associated tissues of fish.We cloned a new IgT molecule from common carp before and we hoped to know its role in Aeromonas hydrophila infection.In this experiment,we deternined the sequence of transcriptome in head kidney and hindgut of common carp after immersion of Aeromonas hydrophilaand then studied the relative expresion of carp IgT and IgM by quantitative PCR and Western-blotting during the immune responses.At last,We detected the IgT positive B cells in tissues of carp by immunohistochemistry.The first part of the experiment is transcriptome sequencing and analysis of head kidney and hindgut of common carp after immersion of Aeromonas hydrophila.Through the transcriptome sequencing,139,751 Unigene were obtained.we used four databases including NR,GO,eggNOG and Swiss-Prot for gene functional annotation and finally acquired 61585,33091,58807,49696sequence annotation in each databases respectively.The percentage of annotation Unigene were among 23.68%?44.07%.The analysis of transcriptome sequencing data of head kidney and hindgut of common carp after immersion in Aeromonas hydrophila for 3 days and 7 days is to acquire the immune related genes with differential expression.The GO and KEGG analysis of unigene showed that the biological pathways involved in these two tissues are different.Complement and lectin pathway was significantly activated pathway in head kidney,the other activated pathway includes Toll like receptor,NF-?B and TNF associated signal pathways.While in the hindgut,antigen processing and presentation pathway was most active,The activation was varied in hindgut tissues of different sampling time.The cell adhesion pathway was also activated during the infetion.We further analyzed the unigene matched with immunoglobulins in head kidney and hindgut transcriptome.The results showed that both IgM and IgZ homologous sequences(zebrafish IgZ homology)exist in head kidney,while only homologous sequence of IgZ in hindgut.Furthermore,more variable region of immunoglobulin homologous sequences appeared in hindgut than head kidney,it wasin accordance with the demands for recognition antigens.Head kidney and hindgut transcriptome study of common carp provide basic data for further research.Analysis of transcriptome sequencing has shown Ig expression were different in carp head kidney and gut tissues,including its antigen binding capacity and signal pathways,therefore,in order to further deter mine the responses of carp IgT,quantitative experiment are carried out by fluorescence quantitative PCR assay and Western-blotting.The quantitative PCR results showed that,the expression of IgM and IgT both changed in head kidney,spleen,gut and liver,but their expression level are varied.IgT has a stronger response in gut,especially in hindgut of carp after immersion of Aeromonas hydrophila.The multiplication is nearly 50.On this point of view,IgT is the the major antibody in gut associated mucosal tissues in the specific immune response.Secondly,when we compared the expression of these two immunoglobulins,we found that the constitutive expression of IgM was higher,the absolute amount of IgM was great although its multiplication was not obvious.We prepared monoclonal antibody against IgT-CH3 fragment,and used it for western-blotting.The results showed that IgT and IgM positive cells could be activatedin different way.IgM might be a kind of immunoglobulin which had basic expression and hindgut was an important site for immune response of specific IgT.The last part of this experiment is to detect the distribution of IgT positive B cells in head kidney,spleen and hindgut.The results showed that,IgT positive B cells existed in both head kidney and spleen,and the density of the positive signal were more obvious in tissue sections of carp suffered immune stimulus.Enzyme stain showed that there were IgT positive cells both in MMCs and outside of MMCs.In hindgut,IgT positive cells distributed both in epithelium and mucosa la mina propria.But the IgT in mucosa la mina propria appeared late,on account of the recruitment of IgT positive cells from sites outside of mucosa la mina propria.Above all,the function of carp immunoglobulin is very important both in systemic immunity and mucosal immunity.IgT,which has more stronger reactivity in gut of carp,can be produced to resist against Aeromonas hydrophila and it has different characteristic from IgM in immune response.The study of IgT will help us to understand the mechanism of specific immunity mediated by antibody and the differentiation of B lymphocytes of fish.