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A kind of separation and purification method of leech polypeptide

A technology for separation and purification of leech polypeptides, applied in the field of medicine, can solve the problems of separation and purification methods for inactive leech polypeptides, difficulty in screening leech polypeptide activity, etc.

Inactive Publication Date: 2018-10-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of leech polypeptides and the difficulty of activity screening, there is currently no simple method for the separation and purification of active leech polypeptides

Method used

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  • A kind of separation and purification method of leech polypeptide
  • A kind of separation and purification method of leech polypeptide
  • A kind of separation and purification method of leech polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the extraction of leech extract

[0030] Take 1 kg of dried leeches and crush them into 60-mesh powder, add 10 kg of distilled water, and homogenate; take the homogenate, adjust the pH to 8.5, add trypsin at a ratio of 10,000 U of enzyme per 1 g of dried leeches, control the temperature at 50°C, and perform enzymatic hydrolysis After 6 hours, cool to room temperature; after the enzymolysis, add 4 times the volume of ethanol to the above enzymolysis solution, refrigerate and precipitate for 8 hours, filter, and dry the filtrate under reduced pressure to obtain 100 g of leech extract. (Or use the method of ZL200810138936.5 to obtain leech extract)

Embodiment 2

[0031] Embodiment 2: Separation and purification of leech polypeptide

[0032] Human colorectal adenocarcinoma Caco-2 cells were inoculated into transwell chambers, using DMEM medium containing 20% ​​FBS, 100U / mL penicillin, 100μg / mL streptomycin, 2mmol / L L-glutamine, and the culture conditions were: 5%CO 2 , 37°C, the seeding density was 450,000 cells / well, the medium was changed 3 times a week, and the culture was continued for two weeks. The integrity of the Caco-2 cell layer is determined by measuring the TEER value of the cell resistance meter, and the TEER value is greater than 500Ω / cm 2 Can be used for experimental research.

[0033] The leech extract extracted in Example 1 was dissolved in PBS (pH 7.2) buffer solution to prepare a leech extract solution with a concentration of 1 mg / mL. Before the experiment started, the culture medium was removed from the transwell chamber, and the culture chamber was washed twice with PBS. Add 0.3 mL of leech extract solution to eac...

Embodiment 3

[0036] Example 3: Purification of leech extract without using Caco-2 cells was separated and purified by Q Sephrose anion exchange chromatography column

[0037] The leech extract extracted in Example 1 was dissolved in PBS buffer to prepare a leech extract solution with a concentration of 1 mg / mL. After Q Sephrose Fast Flow anion exchange column chromatography, 2mol / L NaCl solution was eluted linearly, and the elution curve is shown in the appendix Figure 4 : Separation results of leech extract not purified by Caco-2 cells by Q Sephrose Fast Flow anion exchange chromatography column.

[0038] From attached Figure 4 And attached figure 2 It can be seen from the comparison that after purification by Caco-2 cells, the isolated components of the leech extract are significantly reduced, which is beneficial to the later separation and purification.

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Abstract

The invention discloses a method for separating and purifying leech polypeptides. First, leech extracts are added to a system imitating the absorption of human intestinal cells for cultivation, and then the absorbed substances are chromatographically separated. The inventors of the present invention have solved the problem of separation and purification of leech polypeptides from different angles. By first purifying the leech extract with Caco-2 cells, the digestion and absorption process of the traditional Chinese medicine leech in the body has been imitated. Absorption, a large number of polypeptides that cannot be absorbed by human intestinal cells are first separated, which simplifies the separation and purification process, and then can be separated and purified by anion exchange column chromatography and gel filtration chromatography to be absorbed and utilized by the human body of polypeptides. The polypeptide isolated and purified by the method of the invention has the effect of inhibiting the migration of vascular smooth muscle cells.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a method for separating and purifying leech polypeptides. Background technique [0002] Atherosclerosis is the thickening and hardening of the arterial wall due to the accumulation of lipids in the arterial wall, which in turn causes the artery to lose its elasticity and narrow the lumen. It is a chronic inflammatory disease involving a variety of cells and cytokines. With the deepening of the research on the process of atherosclerosis, more and more inflammatory factors such as the interaction between atherosclerosis-related cells and the interaction between cytokines and cells have been studied. The cells involved in atherosclerosis mainly include: vascular endothelial cells, vascular smooth muscle cells, and immune cells, among which immune cells are mainly macrophages, in addition to neutrophils, mast cells, dendritic cells, and T cells. [0003] The proliferation and mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/18A61K38/17A61P9/10
CPCA61K38/00C07K14/43504
Inventor 宋淑亮吉爱国成龙
Owner SHANDONG UNIV
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