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Novel self-assembled collagen and its preparation method

A collagen and self-assembly technology, applied in the field of protein engineering, can solve the problems of the preparation method of collagen samples and the complex self-assembly process of collagen

Active Publication Date: 2021-06-15
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The self-assembly process of collagen is very complicated. Most of the current research focuses on the influence of collagen concentration, self-assembly time, solution pH, electrolyte, substrate and other factors on the collagen self-assembly process.
[0005] Therefore, the preparation method of self-assembled procollagen samples still needs to be improved

Method used

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  • Novel self-assembled collagen and its preparation method
  • Novel self-assembled collagen and its preparation method
  • Novel self-assembled collagen and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Preparation of self-assembled collagen, taking the preparation of self-assembled bovine type I collagen as an example.

[0034] Self-assembled collagen was prepared by using 5 mg / ml bovine type I collagen aqueous solution as raw material.

[0035] (1) The concentration of collagen is 8000 U / g according to E / S. First, trypsin (Trypsin) is added and hydrolyzed at 37°C for 3 hours. During the hydrolysis, the pH is adjusted with 0.1mol / L NaOH to maintain the pH of the reaction system at a constant value of 8.0. Inactivate the enzyme in a boiling water bath for 20 minutes; add alkaline protease (Alcalase) to continue hydrolysis, and hydrolyze at 55°C for 3 hours. cool down. Finally, centrifuge at 5000g / min for 10min, discard the precipitate, and collect the supernatant.

[0036] (2) Ultrafiltration is performed on the hydrolyzate obtained in step (1) with a 1 kDa ultrafiltration membrane to obtain a collagen polypeptide solution, which is placed in a refrigerator at 4° C. ...

Embodiment 2

[0041] Preparation of self-assembled collagen, taking the preparation of self-assembled fish skin type I collagen as an example.

[0042] Self-assembled collagen was prepared by using 5mg / ml fish skin type I collagen aqueous solution as raw material.

[0043] (1) The concentration of collagen is 8000U / g according to E / S. First, add trypsin (Trypsin) and hydrolyze at 37°C for 6 hours. During the hydrolysis, use 0.1mol / L NaOH to adjust the pH to keep the pH of the reaction system at a constant value of 8.0. Inactivate the enzyme in a boiling water bath for 20 minutes; add alkaline protease (Alcalase) to continue hydrolysis, and hydrolyze at 55°C for 6 hours. During the hydrolysis, use 0.1mol / L NaOH to adjust the pH to keep the pH of the reaction system at a constant value of 8.5; cool down. Finally, centrifuge at 5000g / min for 10min, discard the precipitate, and collect the supernatant.

[0044] (2) Ultrafiltration is performed on the hydrolyzate obtained in step (1) with a 3k...

Embodiment 3

[0047] Example 3 Electrophoresis analysis of the obtained collagen polypeptide and self-assembled collagen polypeptide

[0048] Select the collagen polypeptide solution obtained in Example 1 and the collagen polypeptide solution after self-installation to carry out electrophoresis analysis, specifically as follows:

[0049] SDS-PAGE gel electrophoresis analysis

[0050] The bovine type I collagen polypeptide before and after self-assembly obtained in Example 1 was identified by SDS-PAGE gel electrophoresis at 5% stacking gel concentration, 20% and 12% separating gel concentration.

[0051] The result is as Figure 1a As shown, comparing collagen with standard protein Maker, it can be seen that the molecular weight of bovine collagen polypeptide after hydrolysis becomes smaller, and most of them are concentrated at about 6.8kDa. S in the figure represents the bovine type I after protease hydrolysis in Example 1 Collagen peptides.

[0052] The result is as Figure 1b As shown...

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Abstract

The invention discloses self-assembled collagen and its preparation method and application, wherein the preparation method of self-assembled collagen comprises the following steps: using trypsin and alkaline protease to enzymolyze animal-source collagen so as to obtain The enzymatic hydrolysis product; the enzymatic hydrolysis product is subjected to ultrafiltration to obtain a collagen polypeptide solution; the collagen polypeptide solution is mixed with acetic acid and then dialyzed to obtain the self-assembled collagen, wherein the Dialysis uses phosphate buffered saline as the external fluid for dialysis. This method uses specific proteases to hydrolyze collagen, not only to obtain low-molecular-weight collagen peptide solutions, but also to generate specific "sticky ends" at the ends of the peptides, which can better support organs and protect the body in vivo through self-assembly. of collagen fibers.

Description

technical field [0001] The present invention relates to the field of protein engineering, specifically, the present invention relates to the field of self-assembly of polypeptide molecules, more specifically, the present invention relates to self-assembled collagen and its preparation method and application. Background technique [0002] Collagen is a natural protein synthesized by fibroblasts in animals. It is a structural protein of the extracellular matrix, also known as collagen. Good mechanical structure, thus playing a role in supporting organs and protecting the body. The (Gly-X-Y)n repeating amino acid sequence unit is the molecular basis of the triple helix structure of collagen, in which glycine (Gly) occupies the axial position of the triple helix, and proline and hydroxyproline are high frequency in the X and Y positions respectively. Emerges and plays an important role in the stability of the triple helix. Collagen and its degradation products are rich in natu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C07K1/34
Inventor 裴雪涛南雪游子娟单紫筠魏红姚海雷贾茜媛龙冬莹
Owner ACADEMY OF MILITARY MEDICAL SCI
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