A new method to induce myasthenia gravis models and the protective effect of soluble decay accelerating factors

Abstract

ABSTRACT. It is expensive to induce experimental autoimmune myasthenia gravis (EAMG) by active immunity, and difficult to obtain natural acetylcholine receptor (AChR). We sought a new method of inducing EAMG by immunizing rats with artificially synthesized AChR. The AChR mRNA in TE671 cells was extracted and reverse transcribed. The inclusion body was purified and protein concentration was determined, and the EAMG animal model was used for induction. The serum was extracted from rat blood. The antibody titer was determined using enzyme-linked immunosorbant assay (ELISA). The concentration of decay accelerating factor (DAF) in the rat serum was determined by ELISA, and the metabolism of serum rDAF was determined by western blot. We evaluated the inhibition of rDAF by determining the 50% complement hemolysis unit in the rat serum. The extracellular domain (ECD) nucleotide sequence clone produced by polymerase chain reaction was completely consistent with that in the human gene bank; it was induced by isopropyl β-D-1-thiogalactopyranoside to express the protein after insertion into vector pET16b. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the inclusion body protein was the exact target. The ECD protein was able to bind with mAb35 after dialysis and renaturation, which demonstrated protein activity. The soluble ECD protein was used to immunize rats and obtain the EAMG models. The inhibitory effect of the complement was unsatisfactory owing to high decay rate after rDAF injection into the EAMG models. It is easy to induce the EAMG model by obtaining the AChRTEα1 subunit ECD protein using the substitution method.

Animal models of myasthenia gravis: utility and limitations

“… 32 Regulatory proteins that inhibit MAC formation (such as MIRL-CD59 that inhibits MAC assembly) and control the activation of the complement cascade (such as the decay-accelerating factor or CD55, which inactivates C3 and C5 convertase enzymes) represent crucial players in EAMG development, and their modulation has been a target of recent experimental approaches aimed at complement depletion. 117 , 120 – 123 Pharmacological inhibition of the complement activation pathway is an alternative strategy to depleting approaches. Indeed, the administration of rEV576, a specific C5 complement component inhibitor, is able to reduce the severity of passive transfer of EAMG and the progression of acute experimental MG, reducing C9 deposits at the NMJ.…”