Development And Application Of Monoclonal Antibody Against IgM Of Chinese Sucker(Myxocyprinus Asiaticus)

Chinese sucker(Myxocyprinus asiaticus),also known as Zibian,Muyepan,Huopai,which are mainly distributed in the Yangtze and Minjiang River system.Chinese sucker belongs to the family Catostomidae,which is a freshwater species of high ornamental and economic value because of its beautiful appearance and delicious meat.However,the number of Chinese sucker has been declining gradually due to over-fishing and water pollution.With the enlargement aquaculture scale,diseases have been also increasing gradually,which not only results in huge financial losses for the fisherman,but also restricts the healthy development of aquaculture.Immunotherapy and prevention strategies based on the fish immune system and its mechanisms of action are increasingly valued for their safety and effectiveness.As the lowest vertebrates,fish have both non-specific immunity and specific immunity.Specific immunity system is the most important mechanisim for organism fighting with pathogen.The remarkable feature of specific immunity system is that have astonishing of immunologlobulins.There are three isotypes of Igs in teleosts:IgM,IgD and IgZ/IgT.IgM is the earliest antibody in adaptive humoral immunity and the"head force"of anti-infection.Therefore,studying the structure and fuction of IgM,the expression level of IgM protein in tissues and the immune response of serum IgM of Chinese sucker can not only provide theoretical basis for the immune prevention and formulation of treatment strategies in the Chinese sucker,but also enrich the understanding of immune mechanism of teleosts.The research in this paper mainly includes the following aspects:1)The 5-6 weeks old Balb/c female mice were immuzed by the IgM of Chinese sucker,which was purified by Protein A affinity chromatography.One hybridoma secreting monoclonal antibody against Chinese sucker IgM was obtained by the cell fusion,named as 1-C6A12.The monoclonal antibody was screened and characterized by indirect enzyme-linked immunosorbent assay(ELISA)and Western blotting.The results of specificity showed that the 1-C6A12 was specific for Chinese sucker serum IgM and did not recognize the serum IgMs of other four fish species.2)The expression level of IgM protein in different tissues and body fluids of Chinese sucker was analyzed by the 1-C6A12.The results of Western blotting demonstrated that the IgM protein of Chinese sucker was expressed in kidney,spleen,head kidney,gill,intestine,skin,liver,heart,peritoneal cells,peripheral blood lymphocytes,serum,peritoneal fluid,intestinal mucus,bile and body surface mucus.And its expression level was highest in serum,higher in peritoneal fluid,kidney,spleen,head kidney,gill and intestine,lower in intestinal mucus,bile,body surface mucus,skin,peritoneal cells and peripheral blood lymphocytes,extremely low in liver and heart,and IgM protein is not detected in muscle,brain and gonads.The results of indirect ELISA showed that the expression level of IgM protein in five body fluids of Chinese sucker was serum>peritoneal fluid>intestinal mucus>body surface mucus>bile,which was consistent with the results of Western blotting.3)The 1-C6A12 was used to detect the percentage of mIgM~+lymphocytes of Chinese sucker.Indirect immunofluorescence assay(IFA)analysis suggested that the1-C6A12 could recognize mIgM molecule of Chinese sucker.The 1-C6A12 was used to identify mIgM~+lymphocytes in peripheral blood,head kidney,peritoneal cavity and spleen by flow cytometric,and the results exhibited that the percentages were 15.9%,10.7%,4.32%and 4.16%,respectively.4)The 1-C6A12 was used to explore the phagocytic function of mIgM~+lymphocytes of Chinese sucker.IFA and Flow cytometric analysis showed that mIgM~+lymphocytes in spleen,peritoneal cavity,head kidney and peripheral blood of Chinese sucker had phagocytic function,and the percentage were 2.06%,1.95%,1.42%,0.976%,respectively.5)The immune response of serum IgM of Chinese sucker immunized with Aeromonas hydrophila was investigated using TAS-ELISA based on biotinylated1-C6A12.Chinese sucker were immunized with Aeromonas hydrophila by intraperitoneal injection and immersion,respectively.TAS-ELISA showed that the IgM titer of immersion group and intraperitoneal injection group were significantly higher than in the control group.The serum IgM titer of 14-30d after the initial immunization in the immersion group was 2~5,and reached a peak value of 2~6 during 41-64d.In the intraperitoneal injection group the serum IgM titer increased at 14-30d and 41-48d after the initial immunization,and reached a peak value of 2~9 during 48-64d.In this study,monoclonal antibody against Chinese sucker IgM was produced by immunizing mice with the purified IgM from serum.The 1-C6A12 was screened and characterized by indirect ELISA and Western blotting.Furthermore,the expression level of IgM protein in different tissues and body fluids of Chinese sucker,the percentages of Chinese sucker mIgM~+lymphocytes in peripheral blood,head kidney,peritoneal cavity and spleen and their phagocytic ability were analyzed,and the immune response of serum IgM of Chinese sucker immunized by Aeromonas hydrophila was investigated.This monoclonal antibody provides a powerful tool for studying the Chinese sucker immune system.