Agglutination Activities Of Haemolymph And Tissue Extracts In Scallop Chlamys Farreri And Development And Characterization Of Monoclonal Antibody To A C-type Lectin

Lectins contribute a lot to the scallop humoral immune system for avoidingthe pathogen invasion and environmental stimulus. They play an important role in thepathogen recognition, as well as the physiological process of hemostasis, coagulation,material transport and wound healing in scallops. In this dissertation, the agglutinationactivities of the cell-free haemolymph (CFH) and haemocyte, gills, mantle,hepatopancreas, gonad, kidney, adductor muscle of C. farreri were tested separatelyagainst the rabbit blood cells (RBC), mouse blood cells (MBC) and the fluoresceinisothiocyanate-labelled microbes, and a mannan-binding lectin (MBL) was purifiedfrom CFH by affinity chromatography.; and then, monoclonal antibodies wereproduced, and they were used to analyzed the CfLec290distribution characteristics ofC. farreri haemocytes as well as other organizations combined with RT-PCR and insitu hybridization.(1) Agglutination activities of haemolymph and tissue extracts in scallop C.farreri were tested. In this dissertation, cell-free haemolymph (CFH) and crudeextracts from the haemocytes, gills, mantle, hepatopancreas, gonad, kidney andadductor muscle of C. farreri were obtained, and their agglutination activities weretested separately against RBC, MBC and fluorescein isothiocyanate-labeled microbes.Agglutination inhibition were carried out with EDTA and saccharides. The resultsshowed the CFH could aggregate Vibrio anguillarum, Staphylococcus aureus,Bacillus subtilis, Pichia pastoris and MBC, and the agglutination could be inhibitedby DEAE dextran, EDTA, D-mannose-6-phosphate (M6P), N-acetyl glucosamine(GlcNAc), mannan, zymosan A, chondroitin sulphate, peptidoglycan (PGN) andlipopolysaccharides (LPS); Gill extracts aggregated S. aureus, B. subtilis and P.pastoris, and the agglutination activity could be inhibited by EDTA, N-acetyl galactosamine (GalNAc), mannan, zymosan A, chondroitin sulphate, PGN and LPS;Mantle, hepatopancreas, gonad and kidney extracts could only aggregate B. subtilis,and this could be inhibited by mannan, zymosan A and PGN; however, noagglutination activity was detected using haemocyte or adductor muscle extracts. Theresults indicated that multiple types of lectins exist in C. farreri, and CFH has thehighest agglutination activity.(2) Mannan-binding lectin (MBL) was purified from haemolymph of C.farreri.MBL was purified from CFH by affinity chromatography using a mannan-sepharosecolumn and analyzed using native-and denaturing-PAGE. The results showed that thepurified MBL could aggregate Vibrio anguillarum, and exhibited a molecular mass of645kDa using native-PAGE and73kDa using SDS-PAGE, suggesting that C. farreriMBL is an polymer composed of one subunit.(3) Production of the monoclonal antibodies against rCfLec290. Therecombinant protein rCfLec290were produced and purified in vitro. And then thepurified rCfLec290was used to immunize the mouse, and after the cell fusion, ELISAand western blotting were employed to screen the fusion cells, Finally,3hybridomaswere achieved, MAb1A2,1A7and2B6.(4) Distribution of C-type lectin Cflec290in C. farreri were detected. First,Semi-quantitative RT-PCR and in situ hybridization was employed to detect theCflec290mRNA distribution, and the monoclonal antibodies against rCfLec290wereproduced to detect the distribution of the protein CfLec290in different tissue by IIFAand western blotting. The results showed that: In semi-quantitative RT-PCR, Cflec290mRNA expressed at the highest level in mantle, kidneys and gonads, moderate in gillsand adductor muscles, least in hepatopancreas; In ISH, positive signals were mainlyobserved in the epithelium of mantle and hepatopancreas cells; In IIFA, the positivefluorescent signals were mainly distributed in the membrane of haemocytes, and theedge of gills, mantle, kidney and glands.