Application of transitional scorpion toxin BmK abT
A scorpion toxin and transitional technology, applied in the field of natural active polypeptide - transitional scorpion toxin BmK abT, can solve the problem of undetectable
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Embodiment 1
[0039] Example 1 Pharmacological combination of BmK abT and rat brain synaptosomal membrane
[0040] First, select 5-8 weeks old, 250-350 g adult male Wistar rats, prepare brain synaptosomal membranes according to the method described by Jia Lingyun et al. [Neuroreport 16, 3359-3362 (1999)] Body membrane suspension includes (all mmol / L): choline chloride, 140; calcium chloride, 1.8; potassium chloride, 5.4; magnesium sulfate, 0.8; D-glucose, 10; HEPES-Tris, 25, The pH was adjusted to 7.4 with Tris and immediately used for Biosensor analysis. All buffers in the protocol contained the following protease inhibitors: PMSF (50 μg / ml, Wako Company, Japan); pepstatin A (1 μmol / L, Sigma); iodoacetamide (1 mmol / L, Wako Company, Japan ) and 1,10-phenanthroline (1 mmol / L, Dojindo Company, Japan). The concentration of rat brain synaptosomal membrane was determined by Bio-Rad protein assay with BSA as the standard protein.
[0041] Samples were then fixed and subjected to BIAcore analys...
Embodiment 2
[0044] Example 2 Competitive binding of BmK abT and other scorpion toxins on sodium channels in rat brain synaptosomal membranes
[0045] Same concentration (1.0×10 -5 mol / L) of BmK abT, BmKAS, BmKIT2 and BmKI were incubated with a certain concentration of rat brain synaptosomal membrane (358 μg / ml) at 37°C for 30 minutes, and then flowed through the chip immobilized with BmK abT. Wherein, the mixture solution incubated with BmK abT and synaptosomal membrane was used as a positive control, and the same amount of synaptosomal membrane was injected alone as a negative control. figure 2The inhibitory effects of BmK abT (A), BmK AS (B), BmK I (C) and BmK IT2 (D) on the binding of BmK abT immobilized on the chip to rat brain synaptosomal membranes are shown. The results showed that under this condition, the binding of BmK abT on the chip to the rat brain synaptosomal membrane could be significantly inhibited by the BmK abT incubated with the synaptosomal membrane, and the inhibit...
Embodiment 3
[0047] Example 3 Pharmacological binding of BmK abT to nerve cord synaptosomal membrane of cotton bollworm
[0048] First, the insect nerve cord synaptosomal membrane was prepared from the cotton bollworm according to the method described by Lima et al. [Insect Biochem. 4, 413-422 (1989)]. Cotton bollworm nerve cord synaptosomal membrane suspension includes (all mmol / L): choline chloride, 140; calcium chloride, 1.8; potassium chloride, 5.4; magnesium sulfate, 0.8; D-glucose, 10; HEPES-Tris, 25, the pH value was adjusted to 7.4 with Tris and immediately used for Biosensor analysis. All buffers in the protocol contained the following protease inhibitors: PMSF (50 μg / ml, Wako Company, Japan); pepstatin A (1 μmol / L, Sigma); iodoacetamide (1 mmol / L, Wako Company, Japan ) and 1,10-phenanthroline (1 mmol / L, Dojindo Company, Japan). The concentration of the synaptosomal membrane of the nerve cord of cotton bollworm uses BSA as the standard protein, and the protein concentration is d...
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