Common Carp (cyprinus Carpio L.) Intestinal Mucous Cells Of The Preliminary Study Of Serum Igm Transport Mechanism

Serum IgM was purified from common carp (Cyprinus carpio L.) and then theIgM was used to make a affinity chromatography to purify the IgM receptor on intestineepithelial cell membrane of common carp (fpIgR),Immunize the Guinea pig with fpIgRand then observe the distribution of it.Serum immunoglobulin (Ig) from common carp (Cyprinus carpio L.) waspurified by using ammonium sulfate precipitation followed by SephadexG-200 sizeexclusion chromatograph and MBP affinity purification. The molecule mass of thewhole IgM was 885kDa. Serum IgM was assessed by sodium dodecyl sulphatepolycarylamide gel electrophoresis (SDS-PAGE) and HPLC.The results indicated thatthe serum IgM can be separated into two subunits: the heavy chain and the light chain,with the molecular masses were 82kDa and 29kDa, respectively.The Ig purified by SephadexG-200 was used to make a chromatography foraffinity purification of the receptor on intestine epithelial cell membrane (fpIgR).SDS-PAGE analysis showed that the molecular mass of the receptor was 43kDa.Andthen the fpIgR and the mixture of fpIgR and IgM were analysed by HPLC. The resultsshowed that fpIgR binded IgM.The intestine tissue and lymphocyte in spleen were fluorescence labeleddifferently.Firstly, the intestine tissue was fluorescence labeled by FITC-IgM, the epithelialmucosal cell binded serum IgM and at the same time, the lamina propria mucosae wasalso labeled. Secondly, fpIgR purified by affinity chromatography was used to prepareGuinea Pig antisera (Anti-fpIgR) and then used to label the intestine tissue indirectly, theresults showed that the distribution of fpIgR was accordance with mucosal cell. Lastly,the intestine tissue was blocked by Anti-fpIgR and then was labeled by FITC-IgM, Theresults showed the binding of IgM and intestine tissue was decreased obviously. Sum upall above, the fpIgR might be the key molecule participating in the binding of intestinetissue and IgM, moreover the lamina propria mucosae of intestine tissue was labeledattributing to the dispersed lymph tissue.The lymphocytes in spleen were fluorescence labeled. Firstly, FITC-IgM labelingshowed that lymphocytes in spleen binded IgM, while the erythrocytes did not. Secondly,Anti-fpIgR and FITC-Goat anti Guinea pig whole molecule IgG were used to labellymphocytes in spleen, the results showed that both the lymphocytes and erythrocytescould not be labeled. So the fpIgR did not exist on cells of spleen.