Molecular, Responsive And Functional Characterization Of Immunoglobulin Isotypes From Carassius Auratus

Recently,a unique immunoglobulin isotype had been identified in teleost namedζ(in zebrafish)orτ(in rainbow trout)besides two universal immunoglobulin heavy chain isotypesμandδ.However,in crucian carp(Carassius auratus),the situation was remain mysterious.In present study,five isotypes were identified and characterized in Carassius auratus in total,including two chimeric isotypes(μ-ζ-1 andμ-ζ-2),twoζisotypes(ζ-1 andζ-2)andμ.Among these isotypes,ζ-2 represent an "intra-chimeric" pattern as Cζ3-Cζ4-Cζ3-Cζ4 structure whileμ-ζ-1 andμ-ζ-2 were showed different "inter-chimeric" splicing patterns as Cμ1-Cμ2-Cζ3-Cζ4(μ-ζ-1)and Cμ1-Cζ2-Cζ3-Cζ4(μ-ζ-2).This diversification was first mentioned in early vertebrate evolution.In addition,a unique hinge-like region was noticed between Cμ1 and Cμ2 inμgene as well as a possible Clq binding site located in Cμ3 domain.To comprehend different Igs' positions and responsibilities in immune response, crucian carps were immunized with LPS(a typical TI antigen),BSA(a typical TD antigen)and Aeromonas hydrophila(a pathogenic bacteria for crucian carp), respectively.Skin mucus and serum were collected from body surface and caudal vein weekly while intestine protein was obtained at intervals of two weeks during the following 16 weeks.During these phase,a secondary stimulation was performed after 49 days since the first immunization.Sensitive monitoring of the antibody responses was achieved by ELISA and Real-time PCR methods.The results suggested that most secretary IgZ and IgMZ were detected in mucosa-associated immune system while IgM was expressed in all tissues.This expression pattern was much more like IgX in Xenopus and IgA in mammalians.The location of IgM and IgZ in B lymphocyte surface in crucian carps were also been investigated using polyclonal antibodies after peripheral blood lymphocytes isolated from caudal vein.Three markings of lymphocytes were detected in PBL, including IgM+,IgZ+ and IgM+/IgZ+ lymphocytes.In addion,IgM was demonstrated to be involved in lymphocytes phagocytosis of crucian carps by antibody blocking tests.According to these results,the molecular origin and evolution analysis of immunoglobulin heavy chain constant region gene in vertebrates were performed and a hypothetical evolutionary relationship was constructed.