Conotoxin polypeptide CTx-btg01 and preparation method and application thereof

A technology of conotoxin and polypeptide, which is applied in the field of extraction and preparation of polypeptide protein, can solve the problems of less conotoxin and the like, and achieve the effects of high-efficiency anti-insect activity, small side effects, and easy artificial synthesis.

Inactive Publication Date: 2019-06-28
HAINAN MEDICAL COLLEGE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have found that some conotoxins can inhibit the growth of insect cells, but few conotoxins have been reported that can i

Method used

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  • Conotoxin polypeptide CTx-btg01 and preparation method and application thereof
  • Conotoxin polypeptide CTx-btg01 and preparation method and application thereof
  • Conotoxin polypeptide CTx-btg01 and preparation method and application thereof

Examples

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Embodiment 1

[0049] The preparation of embodiment one conotoxin polypeptide

[0050] According to Embodiment 1 of the present invention, the present invention provides a specific preparation method of conotoxin polypeptide CTx-btg01, comprising the following steps:

[0051] 1. Dissection and toxin extraction

[0052] After smashing the shells of 4 barrel-shaped cone snails produced in Hainan, they were dissected and the poisonous tubes were clipped. After simple washing with ddH2O, they were put into 800 μL pre-cooled extraction buffer (a mixture of 0.1 volume % trifluoroacetic acid, 30 volume % acetonitrile and protease inhibitor cocktail ), cut into small pieces, extrude the venom with tweezers and dissolve it in the extraction buffer, vortex, mix well, centrifuge at 10000g 4°C for 10min, the supernatant is the toxin extract, freeze and dry, and then use 8M urea (urea Dissolve in 0.1M Tris-HCl, pH 8.5) before reconstitution.

[0053] 2. Conotoxin polypeptide enrichment

[0054] Dilute...

Embodiment 2

[0061] Embodiment two conotoxin polypeptide sequence identification

[0062] 1. Determination of toxin polypeptide sequence

[0063] Take the 240 μg mixed polypeptide prepared in Step 2 of the above-mentioned Example 1, and carry out component separation (Table 2) through the SCX-HPLC (Shimadzu) system, and the polypeptide is exchanged with strong cation buffer A (10mM KH 2 PO 4 in 25% ACN, pH 3.5) after dilution, the buffer B contains 500mM potassium chloride on the basis of buffer A, and 0-40% linear binary gradient buffer B is used for separation Separation at a flow rate of 1ml per minute for 10min, react with buffer B at a concentration of 40-90% for 2 minutes, and buffer B at a concentration of 90% for 3 minutes, detect absorbance at 214nm, and collect 10 fractions in total by gradient elution. The collected fractions were dried with a SCANVAC concentrator, redissolved with 0.1% formic acid, desalted with a C18 solid-phase extraction column (Strata-X, Phenomenex), and ...

Embodiment 3

[0071] Example three polypeptide synthesis and renaturation

[0072] Chemical synthesis of conotoxin polypeptides: the complete sequence of conotoxin peptides was synthesized using standard amino acid resin chemical synthesis methods (customized by Shanghai Jier Biochemical Synthesis Co., Ltd.), and the molecular weight of the synthesized peptides was determined using ESI-MS (electrospray ionization mass spectrometry) (eg figure 1 ).

[0073] The primary structure of the chemically synthesized polypeptide is subjected to fractional and fixed-point renaturation, so that the disulfide bonds are formed according to the connection mode of I-III and II-IV to form a disulfide bond connection, so as to restore its active structure in its natural state. The specific refolding method is: use a refolding solution (a mixture containing 0.1M Tris-HCl, 0.1M NaCl, 5mM GSH, and 0.5mM GSSG, pH 7.4) to dissolve the synthesized polypeptide at a mass-volume ratio of 1:10, at 25°C Under the condit...

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Abstract

The invention relates to the field of extraction and preparation of polypeptide protein, in particular to conotoxin polypeptide CTx-btg01 and a preparation method and application thereof. The conotoxin polypeptide CTx-btg01 is composed of 17 pieces of amino acid, and the complete-length sequence of the conotoxin polypeptide is GGCCSHPACGVNHPELC. The conotoxin polypeptide CTx-btg01 is adopted as amedicine active ingredient, and is used for the field of insecticide. The conotoxin polypeptide CTx-btg01 has efficient insect resistant activity, is small in toxicity to mammals, can specifically acton insects and has important application value on development of efficient and safe biological insecticides.

Description

technical field [0001] The invention relates to the field of extraction and preparation of polypeptide proteins, in particular to a conotoxin polypeptide CTx-btg01 and its preparation method and application. Background technique [0002] With the widespread use of many chemical pesticides, the resistance of agricultural pests continues to increase, resulting in increasing toxicity and usage of pesticides, which in turn causes great damage to the entire ecological environment, especially seriously threatening human health. Food health concerns and damage to human health, there is an urgent need for new, efficient, and harmless biopesticides. [0003] Cone snail (Cone snail) mainly grows in tropical seas, belongs to the mollusk phylum, gastropod class, Cono family, and is a beautiful snail that lives on coastal coral reefs and beaches. There are more than 500 species of cone snails in the world, which can be divided into three categories according to their eating habits: inse...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/14C12N15/12A01N37/46A01P7/04
Inventor 高炳淼石琼彭超张俊清易云海卞超
Owner HAINAN MEDICAL COLLEGE
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