Snake venom polypeptide and its preparation method

A technology of snake venom polypeptide and sequence, which is applied in the field of snake venom polypeptide and its preparation, can solve the problem of anti-tumor failure, and achieve good anti-tumor activity, short separation time, and fewer separation steps

Inactive Publication Date: 2006-07-12
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to literature reports, domestic scholars only isolated some nerve growth factors and anti-platelet aggregation polypeptides from Agkistrodon venom, and foreign scholars only isolated a neurotoxin with cytotoxicity from Agkistrodon venom, and Agkistrodon halys venom High-purity anti-tumor active ingredients in snake venom have not been obtained for the time being

Method used

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  • Snake venom polypeptide and its preparation method
  • Snake venom polypeptide and its preparation method
  • Snake venom polypeptide and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0037] 1. Separation and purification

[0038] 1) Fully dissolve 20 mg of Agkistrodon halys venom in 500 μl of ultrapure water, centrifuge at 5,000 rpm for 5 min at 4°C, and take the supernatant for sampling.

[0039] 2) Use the reversed-phase silica gel C18 particles with a diameter of 5 μm and a pore size of 300 Å as a stationary phase filler to make a high performance liquid chromatography C18 reversed-phase preparative column (20 × 300mm), with 0.1% trifluoroacetic acid+acetonitrile as the flow phase, the flow rate is 7ml / min, the detection wavelength is 210nm, and the separation temperature is 20°C. On the HPLC chromatography system, gradient elution is carried out in the range of 0-80%, and the elution time is 120min.

[0040] 4) The automatic collector collects the peaks according to the absorbance value of the detector. After collection, the samples are freeze-dried in a freeze dryer, and stored at -20°C for later use. The result is as Figure 4 .

[0041] 2. Detect...

Embodiment 2

[0045] 1) Fully dissolve 40 mg of Agkistrodon halys venom in 500 μl ultrapure water, centrifuge at 6000 rpm at 4°C for 5 min, and take the supernatant for sampling.

[0046]2) Use reversed-phase silica gel C18 particles with a diameter of 5 μm and a pore size of 300 Å as a stationary phase filler to prepare a high performance liquid chromatography C18 reversed-phase preparative column (20×300 mm). The chromatographic mobile phase is 0.1% trifluoroacetic acid and acetonitrile, the flow rate is 7ml / min, the detection wavelength is 210nm, and the separation temperature is 25°C. On the HPLC chromatography system, 0.1% trifluoroacetic acid+acetonitrile is used as the mobile phase, at 0 Gradient elution was carried out in the range of -80%, and the elution time was 120min. .

[0047] 3) The automatic collector collects according to the peaks according to the absorbance value of the detector. After collection, the samples are freeze-dried in a freeze dryer, and stored at -20°C for l...

Embodiment 3

[0049] 1) 20 mg of the crude venom of Agkistrodon akistrodon was fully dissolved in 500 μl of ultrapure water, centrifuged at 5000 rpm for 5 min at 4°C to remove insoluble substances, and the supernatant was taken for later use.

[0050] 2) Use reversed-phase silica gel C18 particles with a diameter of 5 μm and a pore size of 300 Å as a stationary phase filler to prepare a high performance liquid chromatography C18 reversed-phase preparative column (20×300 mm). The chromatographic mobile phase is 0.1% trifluoroacetic acid and acetonitrile, the flow rate is 7ml / min, and the detection wavelength is 210nm. On the HPLC chromatography system, 0.1% trifluoroacetic acid + acetonitrile is used as the mobile phase, and it is carried out in the range of 0-80%. Gradient elution, the elution time is 120min, and the separation temperature is 28°C.

[0051] 3) The automatic collector collects according to the peaks according to the absorbance value of the detector. After collection, the sam...

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Abstract

The invention relates to a venom polypeptide and it's preparing method in the field of biochemistry and biological medicine. The venom polypeptide has the sequence of (I) and the molecular weight is 7480Da. The preparing method is that it dose separating purify to the cobra-venom by C18 revert column on the HPLC chromatography system with the chromatographic condition: mobile phase 0.1% trifluoroacetic acid and methyl cyanide, the mobile speed 7ml / min, it dose gradient elution on the gradient range 0-80% with the time 0-120min, the test wave length 210nm and the separating temperature 20-28 deg.

Description

field of invention [0001] The invention belongs to the field of biochemistry and biomedicine, and specifically relates to snake venom polypeptide and a preparation method thereof. Background technique [0002] Snake venom is a substance secreted by snake glands of venomous snakes. It has pharmacological effects such as analgesia, hemostasis, inhibition of thrombus formation, and antitumor. Some snake venom preparations have been made and used clinically. However, because snake venom is the most complex type of animal poisons, each snake venom contains at least 20 kinds of active ingredients, mainly including various toxins, enzymes and active peptides, and most of the active ingredients directly enter The body may cause serious side effects in the body, such as cardiotoxicity, neurotoxicity, etc. These side effects are the main reasons for limiting the clinical application of snake venom preparations. [0003] With the rapid development of disciplines such as biochemistry, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/16A61P35/00
Inventor 吴少瑜徐伟李志琴张嘉杰万山河吴曙光
Owner SOUTHERN MEDICAL UNIVERSITY
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