Conotoxin polypeptide κ-CPTx-bt105, and method for preparation thereof and application thereof
Disclosed are a conotoxin polypeptide κ-CPTx-bt105, a method for preparation thereof, and an application thereof. The conotoxin polypeptide of the present invention consists of 16 amino acids, has a molecular weight of 1626.62 daltons, and has the full sequence GICCVDDTCTTHSGCL (SEQ. ID NO. 1).
1. A method for preparing a conotoxin polypeptide κ-CPTx-bt105, which consists of 16 amino acids, has a molecular weight of 1626.62 daltons and a complete amino acid sequence of GICCVDDTCTTHSGCL (SEQ ID NO: 1), characterized in that the method comprises: (1) extraction of conotoxin polypeptides; (2) detection of the conotoxin polypeptides; (3) enrichment of the conotoxin polypeptides; and (4) separation, sequencing of the conotoxin polypeptides and sequence selection of the κ-CPTx-bt105 polypeptide.
2. The method for preparing the conotoxin polypeptide according to claim 1 , characterized in that the extraction of the conotoxin polypeptides comprises removing a venom duct from Conus betulinus , placing the venom duct in a polypeptide extracting solution, mixing and separating by centrifugation, and collecting the supernatant for lyophilization.
3. The method for preparing the conotoxin polypeptide according to claim 2 , wherein the polypeptide extracting solution comprises a deionized solution containing 30% of acetonitrile and 0.1% of trifluoroacetic acid, and a protease inhibitor.
4. The method for preparing the conotoxin polypeptide according to claim 1 , wherein the detection of the conotoxin polypeptides comprises: re-dissolving the extracted conotoxin polypeptides with an 8M urea solution and detecting the protein content and the molecular weight distribution of the conotoxin polypeptides.
5. The method for preparing the conotoxin polypeptide according to claim 1 , wherein the enrichment of the conotoxin polypeptides comprises: subjecting the detected conotoxin polypeptides to a reductive alkylation treatment and then enriching the conotoxin polypeptides by extraction column.
6. The method for preparing the conotoxin polypeptide according to claim 5 , wherein the reductive alkylation treatment comprises: adding dithiothreitol at a final concentration of 10 mM; reacting at 56° C. for 1 hour, followed by cooling to room temperature; adding iodoacetamide at a final concentration of 55 mM, after cooling to room temperature, and then reacting in a darkroom at room temperature for 45 min.
7. The method for preparing the conotoxin polypeptide according to claim 1 , characterized in that the separation, sequencing of the conotoxin polypeptides, and sequence selection of the κ-CPTx-bt105 polypeptide comprises: separating the enriched conotoxin polypeptides by strong cation exchange high performance liquid chromatography, performing mass spectrometry of the polypeptides by nano-high performance liquid chromatography-mass spectrometry, and then conducting data analysis and bioinformatics analysis according to the mass spectrometric data generated by mass spectrometry, to obtain the complete amino acid sequence of the κ-CPTx-bt105 polypeptide.
8. A method of inhibiting electric current of a potassium ion channel in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin polypeptide κ-CPTx-bt105, characterized in that the conotoxin polypeptide consists of 16 amino acids, has a molecular weight of 1626.62 daltons and a complete amino acid sequence of GICCVDDTCTTHSGCL (SEQ ID NO: 1).