Eukaryotic Expression And Functional Characterization Of Fusion Protein Of Single Chain Variable Fragment 637 And Human Serum Albumin

Objective: To express the constructed recombinant gene of single chain variablefragment (ScFv) 637 of antibody against the main immunogenic region (MIR) ofacetylcholine receptor (AChR) in myasthenia gravis (MG) and human serum albumin.After the molecular weight of fusion protein was confirmed, the affinity of fusionprotein to AChR in human intercostals muscles was detected. Methods: Afterresuscitating the Pichia pastoris GS115 strains which bears recombinant geneexpression vector, the strong resistant G-418 of multiple copies transformants wereselected. A single colony was incubated in YPD, BMGY and BMMY culture media toinduce expression in turn. Within inducing expression 3 days, 100% methanol wasadded to BMMY every 24 hours to make the final concentration of 1% in totalculture medium. After centrifuging the supernatant of expression medium at 14000gat room temperature, the fusion protein in supernatant was detected primarily by dotblot. The molecular weight of fusion protein was analyzed by Coomassie-stainedsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The affinityof fusion protein to AChR was detected using indirect immunofluorescencetechnology. Results: The fusion protein was secreted in the supernatant of expressionculture medium. Its molecular weight was about 97.4 KD. There was fluorescencebetween the human intercostals muscle cells. Conclusion: The fusion protein wasexpressed from Pichia pastoris GS115 successfully and it could bind to the AChR inhuman intercostals muscles.