ISOLATION AND CHARACTERIZATION OF SPECIFIC CLONES REACTEDW ITH MY XOBOLUS ROTUNDUS RELATED ANTIGEN FROM A COMBINATORIAL PHAGE DISPLAYED SCFV L IBRARY

Polyclonal and monoclonal antibodies, especially the latter were extensively applied in diagnosis and treatment of fish diseases because of their hihg specificity and affinity.Hybridoma cells are pure sourch of monoclonal antibody (MAb) of desired specificity and present excellent promise in clinical implication.However, the potential have been mostly limited in theory since the establishment of hybridoma technology was made by Kohler&Milstein in 1975 for some intrinsic problematic limitations, especially Human Anti-Mouse Antibody responses(HAMA).In additional, hybridoma technology was labor·extensive and can not be satisfied with requirement of monoclonal antibodies in post-genomic era.The recombinant antibody or engineering antibody technology, especially phage display antibody library, developed in 1990 show great advantage over hybridroma technology in production of monoclonal antibodies.For parasitic diseases caused by Myxozoans, a worldwide constraint factor for development of aquaculture industry and many techniques based on the antibodies were developed to diagnose and studied the immunogenicity and antigenicity of different myxozoan species to find the candidate antigen molecules to control the severe diseases.Significant advances in our understanding of the Myxozoa have been achieved in recent years, however, diagnosis, prophylaxis and therapy of myxospomdiosi8 have not been thoroughly resolved.Myxobolus rotundus Nemezek, 1 9 1 1, is a serious pathogen for the farming of crucian carp, Carassius auratua (L.), allogynogenetic gibel carp and Carassius auratva gibelio(Bloch)in China.Establishment of practical early diagnosis strategy and searching the protective antigen molecules are vital to control the parasitic myxosporean disease.Two strains monoclonal antibodies were previously screened by hybridroma technology, but the low diversity and difficulty of storing limited their application. In the present work, four strains phage displayed monoclonal scFv with better affinity were isolated from previously constructed combinatorial pahge displayed seFv antibody library using M.rotundus related antigen.Their characters were analyzed by ELISA, dot-blot, western·blot, IFAT and immunohistochemi8try.The sequence analysis and alignment displayed that the heavy and light chain of four clones were originated from different mouse germline gene families.Among them, pCAN-6H could specially react with the soluble protein of M.rotundus spores and recognize antigen with a molecular weight of about 34kD.The antigens recognized by pCAN-6H were located in sporoplasm of M.rotundus spores.The pCAN. 3K could specially react with the surface antigen molecules of intact mature M.rotundus spores, especially in the openings of sutural ridge, but no response with polar capsule and sporoplasm.The molecular weight reacted by pCAN-3K ranged from about 17.6kD to 107kD.The pCAN-9A could specially react with the inner membrane of plasmodia.but no response with the middle and outer membrane which indicate probably the heterogenicity of plasmodia membrane and the antigens with molecular weight ranged from about 17.2 kD to 91 kD.The pCAN-3F could react with both antigens of the presporogonic stages and polar capsules which indicate the existence of species·specific antigen for M.rotundus.with molecular weight of about 40kD.