Scorpion venom polypeptide for promoting cell proliferation, preparation method and medicinal application thereof

A technology of cell proliferation and scorpion venom, applied in the biological field, achieves the effect of significantly promoting cell proliferation, single component, and significant effect

Inactive Publication Date: 2011-02-23
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many studies on the cell biological effect and mechanism of action of scorpion venom and its toxin (pure product of a single component) inhi

Method used

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  • Scorpion venom polypeptide for promoting cell proliferation, preparation method and medicinal application thereof
  • Scorpion venom polypeptide for promoting cell proliferation, preparation method and medicinal application thereof
  • Scorpion venom polypeptide for promoting cell proliferation, preparation method and medicinal application thereof

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preparation example Construction

[0037] The preparation method of the scorpion venom polypeptide for promoting cell proliferation according to the present invention is characterized in that it comprises the following steps:

[0038] 1) Collection of East Asian scorpion scorpion venom

[0039] The adult East Asian scorpion is taken from the poisonous biological species breeding base of Jinhua Tiandao Toxic Biotechnology Development Co., Ltd., and the fresh venom is extracted by electric stimulation method, vacuum freeze-dried, and used for later use;

[0040] 2) SVPⅣ separation

[0041] (1) Gel filtration: use molecular sieve chromatography gel Sephadex G-50Medium to separate the crude venom solution of scorpion venom, the size of the chromatography column is 50cm×5.5cm, and the collected peak II is lyophilized and set aside;

[0042] (2) Ion exchange: use CM Sepharose FF filler to further purify peak II through an ion gel column, the chromatographic column size is 100cm×5.5cm, and the eluent is phosphate buf...

Embodiment 1

[0053] Example 1: BmKpp promotes cell proliferation

[0054] The r-h-MCSF-dependent cell line M-NFS-60 was purchased from ATCC (CRL-1838 TM ). M-NFS-60 cells were grown in suspension and routinely cultured in RPMI 1640 medium (penicillin 100∪ / mL, streptomycin 100 μg / mL, HEPES 5958mg / L) containing 10% fetal bovine serum, adding M-CSF 62ng / mL, at 37°C, 5% CO 2 Cultured under conditions, the medium was changed every other day, and the cells in the logarithmic growth phase were taken for experiments. Take the well-growing M-NFS-60 cells, wash them three times with RPMI1640, resuspend with RPMI1640 medium containing 10% fetal bovine serum and adjust the cell density to 8×10 4 cells / ml, inoculate 95 μL of cell suspension in a 96-well plate, add 5 μL of normal saline, different concentrations of BmKpp, and IL-3 after 24 hours of culture, and set 3 parallel wells for each group; after 24 hours or 48 hours of conventional culture, , add 10μLCCK-8 reagent to each well, put the cult...

Embodiment 2

[0056] Example 2: BmKpp promotes cell cycle progression, and cells enter the proliferative phase

[0057] M-NFS60 cells in logarithmic growth phase were taken, washed 3 times with serum-free RPMI 1640 culture medium, added with normal saline (negative control), IL-3 (10ng / mL, positive control) and BmKpp 3μg / mL, for 24 After 1 hour, the cells of each group were taken to make samples, which were used to detect the cell cycle by flow cytometry. The steps of making the specimen are as follows: the concentration of making is 1~5×10 6 / mL cell suspension 0.5 mL, add 2 mL of 70% frozen ethanol, overnight at -4°C; centrifuge at 1000 rpm / min to discard the supernatant; wash twice with PBS; add 50 μL RNase, 450 μL PI dye, and place in the dark at 4°C for 30 min; Cell cycle measurement by cytometry. The results showed that BmKpp significantly decreased the cells in G0 / G1 phase, and significantly increased the proportion of cells in S phase to 49.81% (Table 1). The effect of BmKpp was ...

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Abstract

The invention relates to a scorpion venom polypeptide for promoting cell proliferation, a preparation method and medicinal application thereof. The scorpion venom polypeptide for promoting cell proliferation is characterized in that: the peptide is separated from scorpion venom of Buthus martensii Karsch and purified; and a sequence of 30 amino acid residues at an N end of the scorpion venom polypeptide for promoting cell proliferation is shown as a sequence table SEQ ID No:1, wherein the molecular weight of the corpion venom polypeptide is 7212.32830Da. The scorpion venom polypeptide for promoting cell proliferation is a novel peptide derived from the scorpion venom of the Buthus martensii Karsch, and has the effect of promoting the proliferation of normal or radiated hematopoietic cells. The Buthus martensii Karsch polypeptide (BmKpp) has a single component, precise molecular weight, clear 30 amino acid residues at the N end, obvious effect of promoting cell proliferation, and standard and controllable quality in the process of purification, and thus contributes to scale production. The scorpion venom polypeptide for promoting cell proliferation has the prominent advantages and the wide application prospect in development of medicaments for promoting cell proliferation and preventing cell irritability damage (such as radiation and the like), and can promote colony formation of mouse bone marrow hematopoietic cell after the cell is irradiated by gamma rays or X rays and proliferation of M-NFS-60 cell.

Description

technical field [0001] The invention relates to a scorpion venom polypeptide for promoting cell proliferation, a preparation method and a medicinal application thereof. It is a new polypeptide—Scorpion Venomproliferative Peptide (Scorpion Venomproliferative Peptide from Buthus martensii Karsch, BmKpp), the polypeptide has the effect of promoting the proliferation of hematopoietic cells (normal / after radiation). Belongs to the field of biotechnology. Background technique [0002] There are 6 families, 70 genera, and more than 800 species of scorpions in the world. In China, the East Asian scorpion is the main species. The latter has historically been known both at home and abroad as one of the traditional and precious Chinese medicines in my country. Scorpion venom is a poisonous secretion secreted from the venom sac of the scorpion scorpion. It is the main effective part of the scorpion to treat diseases. Its curative effect on tumors, pain, and cardiovascular diseases is s...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/16A61K38/17C07K1/18A61P7/06
Inventor 董伟华孔天翰王燕郑跃钱邱轶芳王彩霞蒋丽苑李婷邢百倩
Owner 广州医学院
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