Construction Of Eukaryotic Expression Vector Of AChR-IgG Fc Fragment Fusion Protein And Its Expression In CHOk1

Myasthenia gravis (MG) is a disorder of neuromuscular transmission mediated by autoantibodies to the nicotinic acetylcholine receptor (AChR) and involved in activation of complement. It is characterised by fatigable muscle weakness. The most common treatment options used for MG are acetylcholinesterase inhibitors, corticosteroids, immunosuppressants, plasmapheresis, intravenous immunoglobulin, thymectomy, which can relieve the conditions but can not stop course of the disease, and may affect the whole immune system.The pathogenic B cell which produces AChR antibodies, is a key factor in the pathogenesis of MG.Our study is to construct an AChR‐IgG Fc fusion protein which can specificly bind to the FcγRⅡB of pathogenic B cells or FcγR of mononuclear macrophage to inhibit the activation of pathogenic B cell and accelerate its apoptosis.The genomic fragment Hα1-121 of the human skeletal muscle Acetylcholine receptor (AChR)α1 subunit was amplified by PCR and inserted into the eukaryotic expression vector pAN1782.The recombinant plasmid was transfected into CHOk1 cells with screening culture by G418 for stable expression.The expression of the AChR‐IgG Fc fusion protein was detected by Western blot.A 428 bp band was amplified as expected and the sequencing results showed no mutation or frame shift .The recombinant plasmid was confirmed by enzyme digestion and transfected into CHOk1 cells.The stable expression of the AChR‐IgG Fc fusion protein was demonstrated by Western blot .To conclude, the eukaryotic expression vector of AChR‐IgG Fc/pAN1782 was successfully constructed and the AChR‐IgG Fc fusion protein was stably expressed in CHOk1 cells,providing basis for the B cell targeted therapy of myasthenia gravis .Experimental autoimmune myasthenia gravis (EAMG) in mouse is the animal model of MG and helps a lot for finding the pathogenesis and therapy of the disease. The ECD (N-terminal extracellular domain (amino acids 1-210) of theαsubunit of AChR) protein was used to immunize the female Lewis rats and the clinical manifestation , the change of body weight were evaluated. The blood serum of the vena caudalis was extracted every week to test the titer of AChR-ab by ELISA.This method is simpler with a lower cost compared with the traditional EAMG models. The EAMG model can be used to detect the function of the therapeutic protein and research the pathogenesis of MG..