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Preparation method and anti-tumor application of sea anemone polypeptide toxin

A sea anemone polypeptide and toxin technology, applied in the preparation method of peptides, anti-tumor drugs, applications, etc., can solve the problems of colon cancer cell HCT-116 apoptosis that have not been explained, and achieve clear electrophoretic bands and high-efficiency anti-tumor activity , the effect of simple operation

Active Publication Date: 2021-11-23
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This invention needs to use more chemical reagents and biological reagents for preparation, and the obtained purple spot sea anemone polypeptide does not show that it has the effect of promoting the apoptosis of colon cancer cell HCT-116

Method used

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  • Preparation method and anti-tumor application of sea anemone polypeptide toxin
  • Preparation method and anti-tumor application of sea anemone polypeptide toxin
  • Preparation method and anti-tumor application of sea anemone polypeptide toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The extraction of the crude venom of embodiment 1 sea anemone

[0055] 1. Extraction process

[0056] (1) After a 48-h starvation period, the crude venom of sea anemones was extracted by manual extrusion and electrical stimulation.

[0057] (2) Manual squeeze method: clean the live sea anemone with water, put it into a sealed bag and manually rub it up and down 5 times, stimulating the sea anemone for 3 minutes each time to induce its nematocyst to release crude venom.

[0058] (3) Electrical stimulation method: put the live sea anemone in a beaker, clean it with tweezers to remove pollutants, and remove the coelenteric fluid discharged during this process. Then soak the sea anemone in a beaker equipped with artificial seawater, the volume ratio of live sea anemone to artificial seawater is 1:0.5~1.5, the preferred live sea anemone and artificial seawater volume ratio is 1:1 in this embodiment, and use It is electrically stimulated by two carbon electrodes, the voltag...

Embodiment 2

[0064] Example 2 Separation of sea anemone crude venom and detection of cell activity

[0065] 1. Separation method

[0066] The sea anemone crude venom was sequentially passed through 30kD, 10kD and 3kD ultrafiltration tubes with centrifugation parameters of 3500-4500 r / min for 3-5 hours. In this embodiment, the preferred rotation speed was 4000r / min and the time was 4 hours for centrifugation to obtain 4 The fractions are 3kD filter fraction, 3kD cut-off 10kD filter fraction, 10kD cut-off 30kD filter fraction and 30kD cut-off fraction, respectively.

[0067] 2. CCK-8 method was used to detect the inhibitory activity of sea anemone polypeptide on the proliferation of HCT-116 cells

[0068] The colon cancer cell HCT-116 cell culture medium is composed of DMEM basal medium, 10% (v / v) fetal bovine serum and 1% by weight penicillin and streptomycin mixed solution, at 37°C, CO2 concentration is 5% cultured in a cell culture incubator. Collect cells in the logarithmic growth pha...

Embodiment 3

[0074] Example 3 Purification and detection of sea anemone crude venom

[0075] 1. Purification steps

[0076] (1) The 3kD filtered fraction was filtered through a 0.45 μm microporous membrane, and separated by reversed-phase high-performance liquid chromatography. The chromatographic conditions are that the detection wavelength is 210-220nm, the reverse-phase chromatographic column is a C18 semi-preparative column, the eluent A liquid is a 0.08-0.12% volume percent trifluoroacetic acid aqueous solution, B liquid is acetonitrile, and the flow rate is 0.8-1.2 mL / min , the preferred detection wavelength of this embodiment is 214nm, the reversed-phase chromatographic column is a C18 semi-preparative column, the eluent A solution is 0.1% trifluoroacetic acid aqueous solution by volume percentage, B solution is acetonitrile, the flow rate is 1.0mL / min, gradient elution for:

[0077] time A(%, ​​V / V) B(%, V / V) 0 100 0 5 100 0 10 86 14 40 58 42 ...

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Abstract

The invention provides a preparation method and application of sea anemone polypeptide toxin, and the preparation method comprises the following steps: 1, extraction of sea anemone crude venom: after live sea anemone is hungry, extracting an aqueous solution containing sea anemone crude toxin by using a manual extrusion method or / and an electrical stimulation method, freeze-drying the aqueous solution containing sea anemone crude toxin, and dialyzing and desalting by using a biological CE membrane dialysis bag; 2, filtering sea anemone crude toxin molecules: centrifuging the extracted sea anemone crude toxin by using an ultrafiltration tube to obtain a filtered component; and 3, purification of the sea anemone toxin: filtering the filtered component obtained in the step 2 through a microfiltration membrane, carrying out gradient elution through a reversed-phase high performance liquid chromatograph to obtain F1-F3 components, collecting the F1 component, and carrying out freeze drying to obtain the purified sea anemone polypeptide toxin, The sea anemone polypeptide toxin prepared by the invention not only can inhibit the activity of colon cancer cells HCT-116, but also can promote the apoptosis of the colon cancer cells HCT-116.

Description

technical field [0001] The invention relates to the technical field of sea anemone, in particular to a preparation method of a purple-spotted sea anemone polypeptide toxin and its anti-tumor application. Background technique [0002] Sea anemones have tentacle stinging cells for predation and defense functions. The venom secreted by them mainly contains polypeptide and protein neurotoxins and cytolysins. So far, more than 100 polypeptide toxins have been isolated and identified from sea anemones. Able to act on various cell membrane ion channels and has the characteristics of cardiotonic drugs and neurotoxins. [0003] Domestic studies on sea anemone polypeptide toxins began in the early 1990s. Studies have shown that sea anemone polypeptides RSAP Ⅰ and RSAP Ⅱ have activity on sodium channels in guinea pig ventricular myocytes, and the effects of the two are just opposite. A new polypeptide toxin AX-1 was isolated and purified from Zhoushan yellow sea anemone, which can not...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/20C07K1/14A61P35/00A01K61/00
CPCC07K14/43504A61P35/00A01K61/00Y02A40/81
Inventor 高炳淼袁琳符金星廖燕玲
Owner HAINAN MEDICAL COLLEGE
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