Method for separating and purifying leech polypeptide

A technology for separating and purifying leech polypeptides, which is applied in the field of medicine and can solve the problems of difficulty in screening leech polypeptide activity, and no method for separating and purifying active leech polypeptides.

Inactive Publication Date: 2016-03-23
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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  • Method for separating and purifying leech polypeptide
  • Method for separating and purifying leech polypeptide
  • Method for separating and purifying leech polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction of leech extract

[0030] Take 1kg of dried leeches and crush them into 60 mesh powder, add 10kg of distilled water, and homogenize; take the homogenate, adjust the pH to 8.5, add trypsin at the ratio of 10000U enzyme per 1g of dried leeches, control the temperature at 50℃, and enzymatically 6h, cool to room temperature; after enzymolysis, add 4 times volume of ethanol to the above enzymolysis solution to precipitate for 8h, filter, and dry the filtrate under reduced pressure to obtain 100g of leech extract. (Or use the method of ZL200810138936.5 to obtain leech extract)

Embodiment 2

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

[0032] Human colorectal adenocarcinoma Caco-2 cells were inoculated into transwell cells, using DMEM medium, containing 20% ​​FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mmol / LL-glutamine, and the culture condition was 5. %CO 2 , 37°C, seeding density of 450,000 cells / well, changing the medium 3 times a week, and culturing for two consecutive weeks. The integrity of the Caco-2 cell layer is determined by measuring the TEER value with a 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 to prepare a leech extract solution with a concentration of 1 mg / mL. Before the experiment, the medium was removed from the transwell chamber, and the culture chamber was washed twice with PBS. 0.3 mL of leech extract solution was added to each upper chamber, and 1.2 mL of PBS buffe...

Embodiment 3

[0036] Example 3: Without using Caco-2 cells to purify the leech extract, it was separated and purified by QSephrose 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 QSephroseFastFlow anion exchange column chromatography, 2mol / L NaCl solution linearly eluted, and the elution curve is shown in the attachment Figure 4 :The result of separation of leech extract without Caco-2 cell purification by QSephroseFastFlow anion exchange chromatography column.

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

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PUM

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Abstract

The invention discloses a method for separating and purifying leech polypeptide. The method comprises the following steps: adding a leech extract into a system capable of simulating human body enterocyte absorption for culturing; performing chromatographic separation on absorbed substances. By adopting the method, the problem of difficulty in separating and purifying the leech polypeptide is solved from different aspects. The leech extract is purified by Caco-2 cells firstly to simulate the digestive absorption process of leech serving as traditional Chinese medicine in a human body, and a large quantity of polypeptides which cannot be absorbed by human body enterocytes are separated out by simulation of the absorption functions of the enterocytes, so that a separating-purifying process is simplified. Then, polypeptides which can be absorbed by the human body can be separated and purified through anion exchange chromatography and gel filtration chromatography. The polypeptides obtained by separation and purification according to the method have the effect of restraining migration of vascular smooth muscle cells.

Description

Technical field [0001] The invention belongs to the field of medicine, and specifically relates to a method for separating and purifying leech polypeptides. Background technique [0002] Atherosclerosis is the thickening and stiffening of arterial walls caused by the accumulation of a large amount of lipids in the arterial vessel walls, which in turn causes the arteries to lose elasticity and narrow the lumen. It is a chronic inflammatory disease involving a variety of cells and cytokines. With the deepening of research on the pathological process of atherosclerosis, inflammatory factors such as the interaction between atherosclerosis-related cells and the interaction between cytokines and cells have been studied more and more. The cells involved in atherosclerosis mainly include: vascular endothelial cells, vascular smooth muscle cells and immune cells. The immune cells are mainly macrophages, in addition to neutrophils, mast cells, dendritic cells and T cells. [0003] The prol...

Claims

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Application Information

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Patent Type & Authority Applications(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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