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キーワード:
Gasterosteus aculeatus; MHC class I; MHC class II; ECL; N-glycosylation; western blot; immunohistology
要旨:
The three-spined stickleback (Gasterosteus aculeatus) is an important model organism for investigations on the maintenance of polymorphism of the major histocompatibility complex (MHC) of vertebrates. Analysis of functional aspects of MHC diversity in stickleback would benefit from the availability of MHC specific reagents. Here we characterize antisera raised against recombinant fusion proteins of stickleback MHC class I alpha and class II alpha and beta. Western blot analysis using recombinant proteins confirmed the specificity of the antisera. In brain and muscle preparations, neither of the MHC types was detectable. High levels of each MHC receptor type were observed in gills and spleen and lower levels in head kidneys. In histological sections of gills, epithelial cells of primary and secondary lamellae stained positive with MHC class I antiserum, while single, scattered cells stained positive for MHC class II. In sections of spleen and head kidney, considerable numbers of cells positive for either MHC type were detected. Molecular weight shift in SDS-PAGE after deglycosylation of MHC class I alpha and class II beta confirmed the predicted glyco-protein character of the molecules. The majority of MHC II alpha was not glycosylated; only a small fraction of MHC II alpha was susceptible to deglycosylation. This suggests differential expression of the two stickleback MHC II alpha genes (Gaac-DAA, Gaac-DBA) only one of which (Gaac-DBA) has a site for N-linked glycosylation.
