| Experimental autoimmune myasthenia gravis (EAMG) has been a media for studying the pathology and treatment of myasthenia gravis (MG), because of similar characteristics between MG patients and EAMG animals. And because of EAMG, the study of MG has had a rapid development. Active and passive immunity induced EAMG model was used often. Some used the serum of MG patients to passively immunize EAMG and use human muscle or Torpedo electric organs acetylcholine receptor (AchR) as an immunogen to actively immunize EAMG. Compared with passive immunization, the characteristics of T and B cell immune response in active immunized EAMG were more consistent with human MG disease. So most of researches were still induced EAMG with active immunization. Torpedo was not easy to get and it was expensive, and during the extraction process it need to use expensive reagents such as a-bungarotoxin.So it was not suitable to induce EAMG. It was also difficult to get human muscle and human muscles AChR would degrade quickly after isolated and extract less. So it is necessary to find a convenient method to induce EAMG.Since natural AChRs were not readily available, we could artificially get the AChRs to immunize animals. Expressed protein of AChR full-length sequence had a big protein molecular weight. It brought a lot of inconvenience to express gene, transform and express protein. In particular, there might have genetic mutation to make the expressed protein not antigenic.Because there were the major immunogen region (MIR), the specific T cells, B-cell epitopes, and AChR binding sites on extracellular domain (ECD), antibodies(Abs) againstthe muscle AChR played a decisive role in MG and the Abs mainly directed against theAChR subunit N-terminal extracellular domain(ECD)(1-210).AfterAbs combinedwith AChR binding sites, AChRs were inactive, and at the same time it stimulated specificT cell to proliferate, complement system to be actived. Then It ultimately lead MGoccurred. So we could use the fragment to induce EAMG animals.Monoclonal antibodies(mAbs) to human muscle(AChRMU) teact with TE671cellsAChR(AChRTE), including mAbs specific for extrajunctional AChR and the mainimmunogenic region(MIR). So it was feasible to use AChRTE to express recombinantAChRMU subunit ECD protein. Our experiment used the technology such as RT-PCR,vector transformation, the expression of proteins in E. coli, liquid chromatography toobtain highly purified ECD protein, and immunize Lewis rats to make EAMG. Then thepurified soluble promote decay factor (rDAF) was injected in intravenous into EAMGmodel to assess its metabolism in plasma and the protective effect of MG.The experimental results were proved that the PCR cloning ECD nucleotide sequenceentirely consistented with sequence in the human gene pool, and it was successfullyinserted into the vector pET16b, then protein was induced by IPTG. It was confirmed thatthe obtained inclusion body protein was the target protein by SDS-PAGE method. Afterdialysis, ECD protein combined with mAb35. That proved that the protein was active.Using the obtained soluble ECD protein to immunize rats, we induced EAMG model.After using rDAF to inject in EAMG model, we found that it decay rapidly and itscomplement inhibition was unsatisfactory.In summary, using the alternative method to get AChRTE1subunit ECD protein toinduce EAMG model, it was simpler. We also found that rDAF in vitro experiment wasable to play a role of inhibition of complement, but did not achieve the same effect in vivoexperiment. |
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