Isolation Of α-neurotoxin From Naja Naja Atra Venom And Its Application In Purification Of NAchR

【Background】Snake venoms are secreted from the snake’s venom glands-apair of specialized exocrine glands, the secretion contains a variety of naturalpolypeptide components, including cardiotoxin, neurotoxin, phospholipaseA2,venom factors and other peptides. Based on starch gel electrophoresis,neurotoxin from Bungarus multicinctus was categorized to alpha-, andbeta-bungarotoxin. The alpha-neurotoxin effects on the postsynaptic membrane,while the beta-neurotoxin on the presynaptic membrane. According to the aminoacid composition of the neurotoxins, alpha-neurotoxin can also be divided intotwo categories.ClassⅠneurotoxin, known as long-chain neurotoxin, has70-74amino acids, including five pairs of disulfide bonds, it has very high affinitywith the skeletal muscle nAChR; Class Ⅱcontains60-62amino acids and fourpairs of disulfide bonds, known as short-chain neurotoxin,its affinity with nAChR is weaker than the class I. The alpha-neurotoxin was usually purifiedfrom the venom of Indian cobra (Naja naja naja), the Thai cobra (Naja najaSiamensis) or Bangladesh cobra (Naja naja Kaouthia), and the latter one’s isoften referred to cobra toxin. As the combining capacity of cobra toxin tonAChR is moderate, so it is usually used to the purification of the nAChR.Chinese cobra (Naja naja atra) is widely distributed in southern China andlarge-scale reared in the snake park, its venom is very cheap. But there is norelated reports whether its alpha-neurotoxin could serve as an affinity ligand inpurification of nAChR. So we tried to isolate and purify the alpha-neurotoxinfrom the venom of Chinese cobra, and obtain a sufficient amount of protein forthe purification of nAChR, and then establish the experimental autoimmunemyastheia gravis（EAMG）animal model.【Objectives】To isolate-neurotoxin from the venom of Naja naja atra, andto make an-NTs-agarose gel by using the purified-neurotoxin as an affinitychromatography（AC） ligand, for purifying nicotinic acetylcholine receptor(nAChR) from rat skeletal muscle.【Methods】 Using gel filtration chromatography to obtain low molecularpeptides in the crude venom, fractions were measured for their ability to inhibitthe binding of125I--Βungarotoxin to nAChR, select the protein with highestactivity for further purifying with cation exchange chromatography to separatethe-NTs Gradually, SDS-PAGE analysis protein molecular weight and purity;Using purified-NTs coupled to NHS-activated agarose gel as a media toabsorb nAChR in rat skeletal muscle membrane extraction, elute the receptorwith0.2mol/L carbachol to the hydroxyapatite chromatography column, thenelute the receptor from the column with250mmol/L phosphate buffer, collectthe elution and assay the125I-Btx binding activity by RIP. 【Results】 Purified-neurotoxin from Naja naja atra venom showed a~7kDa band in SDS-PAGE. The protein could inhibit125I-Btx binding to nAChR,but in a less extent than natural-Βungarotoxin. The affinity gel coupled withthe purified-neurotoxin efficiently absorbed the125I--Βungarotoxin-bindingactivity from crude rat skeletal muscle extract. And this binding activity couldbe competitively eluted from the affinity gel with0.2mol/L carbamylcholine.【Conclusions】The-neurotoxin from Naja naja atra venom could bepurified with a few chromatography steps and used as a ligand for thepurification of nAChR.