| Acetylcholine receptor is a medium of neurotransmitter and also a key material in the process of mutual transmission of chemical and electronical signal at the neuron synaptic knob. The acetylcholine is located at vesicles in synaptic knob of the transmitting neuron. When an action potential arrives at the synaptic knob, the action potential triggers chemical changes that make acetylcholine vesicles fuse with the plasma membrane of the transmitting cells. Then the fused vesicles release their acetylcholine molecules into the synaptic cleft. The released acetylcholine diffuses across the cleft and binds to the acetylcholine receptor on the receiving cell's plasma membrane. The binding of acetylcholine to acetylcholine receptor opens chemical-sensitive ion channels in the receiving cell's membrane. With the channels open, ions can diffuse into the receiving cell and trigger new action potentials. An enzyme, acetylcholine esterase can break down the acetylcholine, and the ion channels are subsequently closed. Generally, there is no acetylcholine receptor antibody existed in the normal human bodies, but acetylcholine receptor antibody could be appeared for some reasons, such as inheritance and autoimmune disease ( myasthenia gravis). The abnormal antibody can bind with the normal acetylcholine receptor of human bodies, and result in producing myasthenia gravis, which weakens the transmission of neuron synaptic cleft with decreasing the amounts of acetylcholine receptor.The structure and function of acetylcholine receptor have been a crux bottleneck for breaking through the mechanism of autoimmune diseases and the possible therapeutic methods. It will be obviously unsatisfied the demands of the research and therapy for myasthenia gravis if acetylcholine receptor is only prepared from biological organisms. It is un-doubted that an ideal choice to obtain the sufficient amounts of the protein is by using gene engineering technology. However, the soluble expression of acetylcholine receptor always is an obstacle because most of expression products from various constructs are the form of insolubility, known as inclosing bodies.In order to investigate soluble expression of functional region of human acetylcholine receptor (hAChR) a-subunit in host strain E. coli, an open reading frame encoding a-subunit 1-205 amino acid residues (01205) of hAChR was amplified by PCR with pUC-AChRazos as the template and inserted into a vector pMAL-c2X which contained a coding region of Escherichia.coli maltose binding protein (MBP) and a cleavage site of Factor Xa protease. The construct containing fusion gene of MBP-hAChRaios was subsequently transferred into E. coli BL21, and a soluble product, MBP-hAChRaios expressed in the host cells E. coli was achieved by the modulation of MBP. The yield of fusion protein is about 25% analyzed by SDS-PAGE, compared to the total proteins of cell lysate. After sonication and centrifugation of cell pellets, the pure protein around 65 kD was obtained by two-steps, amylose affinity purification and ion exchange chromatography. The recovery yield of former step was 25 mg and the latter was 10 mg (40%) from the 0. 5 L culture. Furthermore, the fusion protein can be hydrolyzed by Factor Xa protease, resulting in the production of hAChRaios, which possesses the structural characteristics itself and immunochemical reaction corresponding to the analogous a-subunit of AChR from Torpedo. It is also shown from the results of Western blot analysis and ELISA that both MBP-hAChRa2os and hAChRtt2os have very similar immunoreactivity.
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