| Univalent antibody fragments, single chain variable fragment(ScFv)from antibody against the main immunogenic region(MIR) of acetylcholine receptor(AchR),do not cross-link AchR molecules or bind complement; however, such fragments derived from anti-MIR monoclonal antibodies(mAbs) are able to protect the trceptor against the loss induced by intact anti-MIR mAb.After digested by endonucleases Nco I and Xho I , the ScFvA7 against MIR in mouse AchR was cloned onto vector pHEN2. Then , the recombinant plasmid pHEN2-ScFvA7 gene was transformed into E.coliHB2151 .The gene in the vector pHEN2 bearing a pelb leader and a c-myc tag were expressed of E.coliHB215.The expression was detected by dot blotting assay using a mouse anti-c-myc monoclonal antibody. Molecular weight of protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The ScFvA7 protein was found in periplasmic fraction, but no expression was discovered in culture medium ,or in periplamic fraction and culture mediun of control E.coliHB2151. Molecular weight of protein was found to be approximately 36KD by SDS-PAGE and western blotting. The ScFvA7 protein has been expressed successfully. This provides the feasibility for the further research on its application and therapy. |
