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A kind of centipede polypeptide slp_sstx and its coding gene and application

A centipede and gene technology, applied in centipede polypeptide SLP_SsTx and its coding gene and application field, can solve the problems of centipede bite clinical symptoms and treatment obstacles

Active Publication Date: 2020-05-15
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Research on clinical symptoms and treatment of centipede bites hampered

Method used

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  • A kind of centipede polypeptide slp_sstx and its coding gene and application
  • A kind of centipede polypeptide slp_sstx and its coding gene and application
  • A kind of centipede polypeptide slp_sstx and its coding gene and application

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preparation example Construction

[0045] In the present invention, the chemical synthesis method of the centipede polypeptide SLP_SsTx and the solution conditional folding method preferably include the following steps: according to the amino acid sequence of the mature peptide of the centipede polypeptide SLP_SsTx, use an automatic polypeptide synthesizer (433A, Applied Biosystems) to synthesize its complete sequence. It was desalted and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) reverse column C18 chromatography, and its purity was determined to be greater than 95%. The linear SLP_SsTx was folded under solution conditions by oxidized glutathione (GSSG) to oxidize its disulfide bonds to form a native tertiary conformation. The molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). SLP_SsTx with native tertiary structure was used for functional detection. The centipede polypeptide SLP_SsTx of the present inven...

Embodiment 1

[0054] Cloning of the SLP_SsTx gene of the less spiny centipede polypeptide:

[0055] I. Extraction of total RNA from the venom gland of the less spiny centipede:

[0056] A. Take out the venom gland tissue of Centipede spp., put it into liquid nitrogen for preservation, take out 500 mg of frozen venom gland tissue, add 10 mL of total RNA extraction buffer (Trizol solution, American GIBCOBRL company), and place in a 20 mL glass homogenizer for homogenization 30 minutes.

[0057] Add an equal volume of phenol / chloroform solution, vortex and mix well, let stand at room temperature for 10 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, and discard the precipitate.

[0058] Add an equal volume of isopropanol to the supernatant, place it at -20°C for 10 minutes, centrifuge at 12,000rpm at 4°C for 10 minutes, wash the precipitate with 75% ethanol three times, and dry it in the air. .

[0059] II. Purification of the venom gland mRNA of the less spinous centipede:

[0060...

Embodiment 2

[0113] Separation and purification of centipede polypeptide SLP_SsTx:

[0114] I. Sephadex G-50 Gel Filtration Chromatography:

[0115] Dissolve 200mg of freeze-dried centipede venom in 20ml of 0.1M phosphate buffer (pH 6.0), centrifuge at 12,000rpm for 10 minutes, take the supernatant and use a balanced Sephadex G-50 gel filtration column (26×100cm) for preliminary separation Purify, elute with the same buffer, and collect with an automatic fraction collector, the flow rate is 3ml / tube / 10min, detect the concentration of protein or polypeptide in the collected solution by ultraviolet light at 280nm, combine the fractions with analgesic activity, freeze-dry,- Store at 20°C for later use.

[0116] II. Reverse High Pressure Liquid Chromatography (RP-HPLC):

[0117] Redissolve the active peak obtained by Sephadex G-50 gel filtration chromatography with 2ml 0.1M phosphate buffer (pH6.0), centrifuge at 12000rpm at 4°C for 15 minutes, take the supernatant, and filter it with a 0.45...

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Abstract

The invention relates to a centipede polypeptide SLP_SsTx as well as an encoding gene and application thereof and belongs to the technical field of biomedicine. The invention provides the centipede polypeptide SLP_SsTx; the amino acid complete sequence of the centipede polypeptide SLP_SsTx is shown as SEQ ID NO. 1. The centipede polypeptide SLP_SsTx provided by the invention has functions of inhibiting a KCNQ family channel and mediating a cardiovascular system, a nervous system and a breathing system and can be used for exploring a KCNQ channel probe template, explaining centipede biting clinical symptoms, and further developing a centipede biting alleviating medicine template.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a centipede polypeptide SLP_SsTx and its coding gene and application. Background technique [0002] Centipede is a kind of traditional Chinese medicine in my country. It has the effects of detoxifying and anti-cancer, calming wind and relieving spasm. [0003] S. subspinipes mutilans L. Koch is a similar species of S. subspinipes mutilans L. Koch, which is mainly distributed in China and Japan, and is common in the middle and lower reaches of the Yangtze River in my country. After long-term evolution, the first pair of centipede feet form hooked, sharp pincers with venomous glands at the end and hard chitin on the outside. There are venom glands used to secrete and store venom in the centipede’s chelas. The venom glands are distributed in the form of vesicles, and the surrounding vesicles can be understood as subvenom glands, which is beneficial for the venom gland cells to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/10A61K38/17A61P39/02
CPCA61K38/00C07K14/43504C12N15/1096
Inventor 赖仞杨仕隆罗雷
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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