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A 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 such as the need to improve the preparation method of collagen samples and the complex process of collagen self-assembly.

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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  • A novel self-assembled collagen and its preparation method
  • A novel self-assembled collagen and its preparation method
  • A novel self-assembled collagen and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Preparation of self-assembled collagen, taking the preparation of self-assembled bovine type I collagen as an example.

[0035] Self-assembled collagen was prepared by using 5 mg / ml bovine type I collagen aqueous solution as raw material.

[0036] (1) Add trypsin (Trypsin), alkaline protease (Alcalase) and papain (Papain) sequentially according to the E / S of 8000U / g collagen concentration. Each protease was hydrolyzed for 2 hours and co-hydrolyzed for 6 hours (the hydrolysis time of each protease can be within 2 to 6 hours). During the water bath, 0.1mol / L NaOH was used to adjust the pH to keep the pH of the trypsin hydrolysis system at a constant value of 8.0. The pH of the alkaline protease hydrolysis system is maintained at a constant value of 8.5, and the pH of the papain hydrolysis system is maintained at a constant value of 7.0. At the end of each protease reaction, inactivate the enzyme in a boiling water bath for 20 minutes and cool down. Finally, centrifuge a...

Embodiment 2

[0042] Example 2 Electrophoresis analysis of the obtained collagen polypeptide and self-assembled collagen polypeptide

[0043] 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:

[0044] SDS-PAGE gel electrophoresis analysis

[0045] 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.

[0046] The result is as Figure 1a As shown, comparing the collagen with the standard protein Maker, it can be seen that the molecular weight of the bovine collagen polypeptide after hydrolysis becomes smaller, and there is no band above 4.6kDa, so it can be seen that the molecular weight after hydrolysis is concentrated at 4.6-10kDa. S in the figure represents the bovine type I collagen polypepti...

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Abstract

The invention discloses self-assembled collagen and its preparation method and application, wherein the self-assembled collagen preparation method comprises the following steps: using trypsin, alkaline protease and papain to enzymolyze the animal source collagen, so as to obtain An enzymatic hydrolysis product of a small molecule peptide; 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 treatment uses phosphate buffer 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): C12P21/06C07K14/78C07K1/34
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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