Preparation Of Single-chain Fragment And Human Serum Albumin Fusion Protein And Its Inhibition To Sera Of Patients With Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disease affecting components of the neuromuscular junction and thus disrupting the signal transduction over the postsynaptic membrane. This autoimmune disease is characterized by muscle weakness that fluctuates, worsening with exertion, and improving with rest. The major target, in80%-85%of MG patients, is the muscle acetylcholine receptor (AChR). Single-chain fragment variable (scFv) is consists of variable regions of heavy (VH) and light (VL) chains, which are joined together by a flexible peptide linker. ScFv retained the complete antigen-binding capability, and it could not activate complement without Fc fragment. However, the small molecular size of scFv has the physiological disadvantage of rapid elimination from the body via kidney. ScFv637was an antigen-specific scFv of MG, which composed of amino acids67-76of the a-subunit (e.g. main immunogenic region, MIR) of the human AChR (hAChR). It was able to protect hAChR from the binding of anti-MIR antibodies and MG patient sera, which makes it an alternative candidate for specific immunosuppressive therapy. As well as scFv, scFv637could rapidly clear from blood. Thus, in this study, we tried to prepare a conjugate by linking the scFv with human serum albumin (HSA) to increase its half-life and improve its stability in blood. ScFv could become a potential candidate to specific immunosuppressive therapy of MG.Objective:To prepare single-chain fragment fusion human serum albumin, for prolonging the half-life of scFv and increasing its stability in blood. It can be an agent for immunotherapy of myasthenia gravis.Methods:The genes of scFv637and HSA were conjugated and expressed in Pichia pastoris. After determining its molecular weight in western blot, the affinity of scFv-HSA fusion protein binding to AChR was detected in immunofluorescence staining. The block effects of the fusion protein on the binding to AChR of MG patient sera and its stability in healthy serum were measured in competitive ELISA.Results:The fusion protein was successfully expressed and the size of molecular weight was about89kDa. Immunohistochemical staining showed the fusion protein could bind to AChR in situ at the neuromuscular junction of human intercostal muscle, In12cases of MG patient sera, the inhibition rate of binding of AChR was from2.0%to77.4%. The stability of fusion protein in static healthy sera remained about3days.Conclusion:The fusion protein of scFv-HSA is prepared successfully and it remained the capability to protect AChR. This approach may make the scFv-HSA a potential candidate for specific immunosuppressive therapy of MG...