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Self-assembled collagen and its preparation method

A collagen self-assembly technology, applied in the field of protein engineering, can solve the complex self-assembly process of collagen, and the preparation method of collagen samples needs to be improved.

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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  • Self-assembled collagen and its preparation method
  • Self-assembled collagen and its preparation method
  • Self-assembled collagen and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The self-assembled collagen was prepared to prepare self-assembled cattle type I collagen as an example.

[0034] Self-assembled collagen is prepared in a 5 mg / ml bull type I collagen aqueous solution.

[0035] (1) The collagen concentration is added to the E / S for 8000 u / g to add trypsin, and then the pH is adjusted with 0.1 mol / l NaOH during hydrolysis, and the reaction system is maintained at 8.0, boiling water bath. The enzyme was killed for 20 min, and then papain was added to 50 ° C for 3 hours, and the pH was adjusted with 0.1 mol / L NaOH during hydrolysis to allow the reaction system pH to maintain 7.0. Bulse the hydraulic enzyme for 20 min, cool. Finally, 5000 g / min centrifuge 10min, discard the precipitate, and collected the supernatant.

[0036] (2) The hydrolyzate obtained by step (1) is ultrafined with an ultrafiltration membrane of 1 kDa to obtain a collagen polypeptide solution, and is placed in a refrigerator at 4 ° C.

[0037](3) The collagen pol...

Embodiment 2

[0041] Example 2 An electrophoresis analysis of obtained collagen polypeptides and self-assembled collagen polypeptides

[0042] The collagen polypeptide solution obtained in Example 1 was electrophoresed by electrophoresis with the self-contained collagen polypeptide solution, as follows:

[0043] SDS-PAGE gel electrophoresis analysis

[0044] The self-assembly polypeptide obtained before and after the SDS-PAGE gel electrophoresis was identified using 5% concentrated glue concentration, 20% and 12% separation glue concentration.

[0045] Such as Figure 1A As shown, the collagen is compared to the standard protein Maker, and it can be seen that the molecular weight of the hydrolysis is small, mostly concentrated below 10 kDa, and the schematic is a cow type I collagen after protease hydrolysis in Example 1. Polypeptide.

[0046] Such as Figure 1B As shown, the collagen polypeptide is self-assembled, the molecular weight is significantly large, mainly concentrated 22.5 kDa, 10 kDa ...

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Abstract

The invention discloses self-assembled collagen and a preparation method thereof, wherein the self-assembled collagen preparation method comprises the following steps: using trypsin and papain to enzymolyze animal-source collagen so as to obtain enzymatic hydrolyzate containing small molecular peptides 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 treatment uses Phosphate buffered saline was used 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, in particular, the present invention relates to the field, and more particularly, the present invention relates to self-assembling collagen and its preparation methods and applications. Background technique [0002] Collagen is a natural protein produced by fibrotara synthesis from fibrous cells, which is a structural protein of extracellular matrix, also known as collagen, widely in connective tissue such as bone, skin, tendon such as animals, so that the tissue and organs have A good mechanical mechanical structure, thereby playing the role of the organ and protect the body. (GLY-XY) N Re-Amino Acid Sequence Unit is a molecular basis constituting the collagen tri-helix structure, wherein glycine (GLY) occupies the axial position of the three spirals, proline and hydroxyproline are high frequently in X and Y positions, respectively. The stability of the three spirals appears and plays an im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C07K14/78C07K1/34
CPCC07K1/12C07K1/34C07K1/36C07K2/00C12P21/00C12P21/06
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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