Expression And Assay Of A7 Gene Of Single Chain Variable Fragment Against Acetylcholine Receptor In Myasthenia Gravis In Pichia Pastoris

Objective. To transform single chain variable fragment against acetylcholine receptor in myasthenia gravis into Pichia yeast and to express the protein in the eukaryotic expression system. Methods: The recombinant vector, which was isolated and purified from Ecoli. DH5_a was linerized by endonuclease Sal land then was transformed into Pichia Pastoris GS115 by electroporation. Next, we isolated the genome of GS115 to conduct the insertion condition of the target gene. Then the target gene was induced to express by methanol. The expressed products were detected by dot blotting assay using a mouse anti-c-myc monoclonal antibody. Molecular weight of target protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Results: The sizes of the recombinant vector and the eukaryotic expression vector were found to be approximately 10000bp and 9300bp; and the ScFvA7 gene was inserted into the chromosome of GS115 successfully. Molecular weight of the ScFvA7 protein was found to be approximately 44KD by SDS-PAGE and western blotting. Conclusion: The ScFvA7 protein has been expressed successfully in the eukaryotic expression system. These lay a foundation for the activity and specificity identification of this protein.