The Antibody Preparation And Functional Analysis Of CTLD4-8 Domains Of Mannose Receptor In Blunt Snout Bream(Megalobrama Amblycephala)

Mannose receptor(MR, CD206) is the first member to be found in the mannose receptor family, it can effectively recognize many endogenous and exogenous mannosylated ligands and plays an important role in both immunity and homeostasis. Its ectodomain has eight C-type lectin-like domains(CTLDs), of which CTLD4-8 show high affinity for endogenous and exogenous mannosylated ligands comparable to that of whole MR. MR expression is not macrophage-restricted, and the expression are also found on mesangial cells, endothelial cells within the lymph and liver, retinal pigment epithelial, myoblasts and microglia in the brain. Although the structure and function of mammalian MR have been well characterized, little is known in fish. Moreover, hitherto, there has been no comprehensive report about the distribution of the fish mannose receptor protein during early embryogenesis and in various tissues of juvenile fish. In the present study, we developed the rabbit polyclonal antibody(Ma MR-CTLD4-8) against MR following the process of construction the expression plasmid, prokaryotic expression and protein purification. Using this antibody, we undertook a systematic survey of the expression of MR by immunohistochemistry. Subsequently, we analyzed the interactions between GFP-E.coli and MR, investigated the role of MR in the phagocytosis of pathogens by macrophages and analyzed the sequent immune response. The research results are listed as blow:(1) Target protein was effectively expressed under the optimized conditions. By SDS-PAGE and Western-blot analysis, we detected the correct recombinant protein with the molecular mass of 110 k Da.(2) One healthy Japanese big-ear rabbit was immunized with the purified protein for three times. Polyclonal antiserum was collected and its titer and specificity were detected by ELISA and Western-blot, respectively. The antibody titer was found to be approximately 1:51,200 and can specifically detect the 170~180 k Da mannose receptor isolated from primary macrophages and head kidney lysates by Western-blot, which clarified the antibody specificity and the feasibility for the further study.(3) We undertook a systematic survey of the expression of mannose receptor during early embryogenesis and in various tissues of juvenile fish by using immunohistochemistry. MR expression was first found in the yolk sac at fertilized egg, and became widely expressed at hatching stage. We discovered that there is a relationship between the MR expression and spatio-temporal localization of early macrophages, and deduced that MR may be expressed by early macrophages, not restricted to terminally differentiated macrophages. MR was widely expressed in the blunt snout bream head kidney, body kidney, brain, liver, intestine, gill, muscle and heart, but scarcely expressed in the spleen. Of interest, the distribution of MR in the spleen and head kidney became widespread following infection with fish pathogen Aeromonas hydrophila.(4) MR protein was located on the macrophage surface as well as in intracellular pool by indirectimmunofluorescence staining.(5) MR in blunt snout bream mediated the non-specific recognition and phagocytosis of heat-killed GFP-expressed E.coli by macrophages and triggered the release of inflammatory mediators like ROS, NO and cytokines like IL-1β and TNF-α.