Refolding Expression Of Acetylcholine Receptor And Construction The Method Of Detecting Anti-achr Antibody

Myasthenia gravis (MG) is an autoimmune disease caused by an immunologicalresponse against the acetylcholine receptor (AChR) at the neuromuscular junction.Anti-AChR antibodies induce degradation of the receptor, activation of complementcascade and destruction of the post-synaptic membrane, resulting in a functional reductionof AChR availability.Nicotinic acetylcholine receptor (nAChR) is a member of ligand gated ion channelsuperfamily. It mediates the rapid transformation from chemical signal to electrical signalat the post-synaptic membrane in neuromuscular junction. nAChR whose relativemolecular weight is about290000, is a pentamer contains four homologic subunits inelectric organ of torpedo and skeletal muscle. AChR in torpedo and fetus is α12β1δγ,while in adult is α12β1δε. All the subunits arrange a cylinder-shaped ion channel complexin α1-γ-α1-δ-β1order. Every subunit has a polypeptide chain which is in an order fromN-terminal to C-terminal a big N-terminal extracellular domain (ECD), three transmembrane domains, a big inner cell ring, the fourth transmembrane domain and aC-terminal tail outside the cell. N-terminal in ECD in nAChR has a high affinity withreceptor agonist and competitive antagonist. There are two nonidentity sites whichacetylcholine acted on as a neurotransmitter at the interface between subunit α1, γ andsubunit α1, δ. Otherwise, the binding site of α-Bungarotoxin (α-BTx) and AChR is mainlyat N-terminal in ECD of α1subunit. ECD of α1subunit also contains the mainimmunogenic region (MIR), which is the main site producing the anti-AChR antibody inMG patients. Exogenous injection with nAChR can produce anti-AChR antibody inanimals, and it can make the animal show the symptom of MG. That is a method toestablish experimental autoimmune myasthenia gravis (EAMG) animal model. It ispossible using the nAChR binding the pathogenic antibody in MG to eliminate theantibody and relieve the clinical symptoms.Detecting the anti-AChR antibody in the serum of MG patient by using the radioimmune assay (RIA) is an important criteria in MG diagnoses. The sensibility of thismethod is88%, and there are different positive incidences in different MG subtypes. Theyare71%of the early-onset ocular MG,88%of late-onset ocular MG,89%of theearly-onset generalized MG,98%of the late-onset generalized MG. Otherwise, thespecificity of diagnosing the MG by detecting the anti-AChR antibody is very high,approximately99.9%. So we hope we can establish a stable anti-AChR antibody RIAdetecting method to help the MG clinical diagnosis and improve the efficiency ofdiagnosis.Experimental autoimmune myasthenia gravis (EAMG) is antibody induced T celldepending autoimmune disease happened in mice. And all the immune reaction is againstAChR. EAMG animal model as an important research technique is still an important basein study of our academic field. There are two major materials at present for establishingEAMG model: one is the electric organ of torpedo, the other is denervated muscle ofmammalian. However, the torpedo’s price is quite high and it is hard to acquire at home.The operation of denervation on mouse is not easy and has a small amount of AChRharvest. So we come to use the method of refolding expression of AChR to attain a stable and biological activity protein. We have tried two methods to express AChR. The first isrefolding and expression of the extracellular domain of the human AChR α1by CHO. Thefragment of1-216amino acid at the N-terminal of nAChR α1was obtained from TE761cells by PT-PCR, and was purified by agarose gel electrophoresis. Than the fragment oftarget gene was cloned into expression vector pcDNA3.1. After restriction endonucleasedigestion and DNA sequencing confirmation, the recombinant ECD (extracellular domain)protein expressed plasmid was transfected into CHO cells. The cells were detected ECDgene replication by real time PCR and identified ECD protein expression byradioimmunoassay of125I mark at the6h/12h/24h/36h/48h/60h/72h respectively aftertransfection. The second is expression of extracellular domain of the mouse AChR α1afterhaving three base mutations by bacillus coil. The three base mutations in extracellulardomain of the mouse AChR α1respectively are Val8Glu, Trp149Arg, and Val155Ala.Then synthesize the above-mentioned genes in the company. Insert the synthetic gene tothe pET-22b carrier, and express in a large number in Bl-21bacterial strain. Afteridentifying the correct of protein expression, refold the denatured protein by solutiondilution method.In conclusion, we try to substitute the natural AChR by refolding and expression theprotein in eukaryocyte or prokaryocyte in this experiment. And we want to establish arapid and handy method to detect the anti-AChR antibody in patients’ serum, which canalso improve the efficiency of MG clinical diagnosis.