Characterization Of Expressed Anti-acetylcholine Receptor Single Chain Variable Fragment A7 In Myasthenia Gravis In Pichia Pastoris

Objective: To determine the specificity of expressed single chain variable fragment (ScFv) A7 of antibody against acetylcholine receptor (AchR) in myasthenia gravis in Pichia pastoris. After the molecular weight of target protein was confirmed, the affinity of target protein to AchR in mouse intercostals muscles was detected. Methods: After resuscitating the Pichia pastoris GS115 strains which bear recombinant gene expression vector, the strong resistant G-418 of multiple copies transformants were selected. A single colony was incubated in YPD, BMGY and BMMY culture media to induce expression in turn. Within 3 days of inducing expression, 100 % methanol was added to BMMY every 24 hours to make the final concentration of 1 % in total culture medium. After centrifuging the supernatant of expression medium at 14000 g at room temperature, the target protein in supernatant was detected primarily by dot blot. The molecular weight of expressed protein (ScFvA7) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The affinity of expressed protein to AchR in mouse intercostals muscles was detected using indirect immunofluorescence technology. Results: The target protein was secreted in the supernatant of expression culture medium. Its molecular weight was about 44 KD. There was fluorescence between the mouse intercostals muscle cells. Conclusion: The target protein has been expressed from Pichia pastoris GS115 successfully and it can bind to the AchR in mouse intercostals muscles.