The Function Of IgM On Immune Protection Of Largemouth Bass(micropterus Salmoides) And Cyprinus Carpio Var. Jian

Largemouth bass(Micropterus salmoides)and Cyprinus carpio var.jian are both important freshwater fishes.It is reported that the annual production amount of largemouth bass and carp in China are as high as 450,000 tons and 300,000 tons,respectively.Cyprinid herpesvirus 3(Cy HV-3),also known as Koi herpesvirus(KHV),is a highly contagious and lethal virus.However,the study on KHVD prevention and largemouth bass immunity are still meager,which caused a major loss in production of Cyprinus carpio var.jian and largemouth bass.Studies show that specific immune response mediated by immunoglobulin is triggered with vaccination or viral infection.Teleost immunoglobulin has been proved to be mainly divided into three types,including Ig M,Ig D,and Ig T/Ig Z,and Ig M is the most dominant and abundant subtype.In this study,six positive hybridomas which designated as MM01H,MM04H,MM06H,MM07H,MM08H and MM11H secreting monoclonal antibodies(MAbs)against largemouth bass immunoglobulin M(Ig M)were produced and MM01H was used as a research tool.Western blot analysis showed that the MAb was specific for the heavy chain of largemouth bass Ig M.Indirect immunofluorescence assay(IFA)analysis suggested that MM01H could specifically recognize membrane-bound Ig M(m Ig M)molecule of largemouth bass.Flow cytometric analysis(FCA)exhibited that the percentages of largemouth bass m Ig M~+lymphocytes in peripheral blood,spleen and pronephros were 12.9%,52.7%and 40.2%,respectively.Moreover,we found that the m Ig M~+lymphocytes of largemouth bass could phagocytose Aeromonas veronii,the phagocytic rates of peripheral blood,spleen and pronephros were 13.0%,44.4%.and 24.6%,respectively.During our previous study,KHV ORF148 was inserted into p EGFP-N1 generating p EGFP-ORF148recombinant plasmid.In the present study,it was confirmed that p ORF148-EGFP fusion protein could be successfully expressed in vitro and in vivo by transfection assay and immunofluorescence analysis,respectively.The results of ELISA showed that immunization with p EGFP-ORF148 recombinant plasmid could significantly(P<0.01)improve the tire of specific antibody against KHV.The RT-q PCR revealed that injection with p EGFP-ORF148recombinant plasmid caused 145.30-,131-,52.27-,12.16-,4.19-,3.05-,4.03-fold increase of IFN-a1,TNF-α,IL-1β,Ig M,CXCa,CXCR1,Mx-1 gene expression in head kidney,respectively,when the PBS immunized carps served as control.In spleen,the expression of IFN-a1,CXCa,TNF-αand Ig M from p EGFP-ORF148 recombinant plasmid injected carps was 22.44-,4.63-,6.14-,4.23-fold higher than that of control group.Post challenge with KHV,the survival rates of carps immunized with PBS,p EGFP-N1,p EGFP-ORF148recombinant plasmid were 30%,35%and 85%,respectively,suggesting that immunization with p EGFP-ORF148 recombinant plasmid significantly protected the carps against the KHV infection(P<0.01).In conclusion,in this study,monoclonal antibody against serum Ig M of largemouth bass was used to detect the distribution,proportion of Ig M~+in tissues/cells of largemouth bass,and to confirm the phagocytosis of m Ig M~+B cells.The p EGFP-ORF148 recombinant plasmid was injected into Cyprinus carpio var.jian to verify the function of Ig M on immune protection.All of results demonstrated that the MAb purified could be used as a tool to study m Ig M~+B cells of largemouth bass,and p EGFP-ORF148 recombinant plasmid is a potential candidate of DNA vaccine against KHV.The study improve the cognition of the immune system function in teleost,as well provide a theoretical basis for the immune prevention of fish.