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Barnacle glue protein 20K derived polypeptide with self-assembly characteristic and biomineralization function as well as preparation method and application of barnacle glue protein 20K derived polypeptide

A technology of biomineralization and self-assembly, applied in biochemical equipment and methods, measurement/testing of microorganisms, microorganisms, etc., to achieve the effects of promoting proliferation and osteogenic differentiation, preventing flocculation, and reducing the number of disulfide bonds

Active Publication Date: 2022-06-10
NAT UNIV OF DEFENSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Barnacle glue contains more cysteine, which is difficult to extract from the cured protein glue, and it is easy to form intermolecular dichotomy after heterologous expression of Escherichia coli Large-scale flocculation occurs due to sulfur bonds, which is not conducive to determining its active state and specific biological functions. The present invention provides a barnacle gum protein 20K derivative polypeptide with self-assembly properties and biomineralization functions, its preparation method and application , cp20k-p3 having self-assembly properties and biomineralization functions, its preparation method and application

Method used

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  • Barnacle glue protein 20K derived polypeptide with self-assembly characteristic and biomineralization function as well as preparation method and application of barnacle glue protein 20K derived polypeptide
  • Barnacle glue protein 20K derived polypeptide with self-assembly characteristic and biomineralization function as well as preparation method and application of barnacle glue protein 20K derived polypeptide
  • Barnacle glue protein 20K derived polypeptide with self-assembly characteristic and biomineralization function as well as preparation method and application of barnacle glue protein 20K derived polypeptide

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Experimental program
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Effect test

Embodiment

[0043] The preparation method of the barnacle gum 20K derivative polypeptide with self-assembly properties and biomineralization function comprises the following steps:

[0044] 1) Prepare a DTT solution with a concentration of 60mM to treat the cp20k sample, take 10ul of a certain concentration of cp20k suspension and mix it with 10ul of 60mM DTT, pipette evenly with a pipette gun, then add the corresponding volume of 4×Loading Buffer to mix and put it into PCR Heating in the amplification instrument at 70°C for 10 minutes, after the sample preparation is completed, use the electrophoresis instrument to run the gel, 220V, 120mA, 10min, then adjust the current to 80mA and run for 30min, remove the film and add fixative for 5min, then stain for 5 hours, pour Add 10% acetic acid solution to decolorize at room temperature overnight;

[0045] 2) Synthesis and identification of cp20k-p3 wild type and its mutants: through sequence analysis, cp20k contains a conserved repetitive sequ...

experiment example

[0056] 1 Materials and methods

[0057] 1.1 Materials

[0058] 1.1.1 Peptides and Cells

[0059] The polypeptide is synthesized by chemical synthesis, and the purity is >95% as identified by mass spectrometry. The cells used in the verification of biomineralization function were mouse bone marrow mesenchymal stem cells (mMSCs).

[0060] 1.1.2 Main reagents

[0061] CCK8 Kit, NuPAGE TM 10% Bis-Tris Gel Mini Protein Gel, NuPAGE R LDS SampleBuffer[4×], DTT (Dithiothreitol, dithiothreitol), prestained protein marker, alizarin red dye, hydrochloric acid, acetic acid,.

[0062] 1.2 Method

[0063] 1.2.1 DTT treatment of cp20k to identify the role of cysteine

[0064]Since cp20k contains as many as 17 cysteine ​​residues, complex intermolecular / intramolecular disulfide bonds are formed, resulting in the automatic formation of flocculent precipitates during purification, lyophilization, and refrigeration. To verify this, a DTT solution with a concentration of 60 mM was prepar...

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Abstract

The invention relates to a barnacle glue protein 20K derived polypeptide with a self-assembly characteristic and a biomineralization function as well as a preparation method and application of the barnacle glue protein 20K derived polypeptide. A potential functional fragment cp20k-p3 (p: peptide) is obtained through homologous sequence alignment. Researches show that the polypeptide has a unique self-assembly characteristic, and can significantly promote proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Cysteine in cp20k-p3 is replaced one by one through alanine scanning, four polypeptide mutants are obtained, and it is found through comparison that formation of disulfide bonds has remarkable influences on the self-assembly morphology and the biomineralization function of polypeptide. The invention not only provides a clue for breaking the biological function of cp20k, but also provides a brand new functional material for bone tissue engineering.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, in particular to barnacle gum 20K derivative polypeptides with self-assembly properties and biomineralization functions, its preparation method and application, that is, cp20k-p3 with self-assembly properties and biomineralization functions and Its mutants, its preparation methods and applications. Background technique [0002] Bone defects and fracture nonunions caused by various diseases such as trauma, infection, osteonecrosis and tumors are often seen in orthopedic clinical work, which leads to a huge demand for bone repair materials in clinical practice. Bone transplantation is the main clinical treatment, among which autologous bone transplantation has the best effect, but its source is very rare and will cause new trauma to patients; allogeneic bone transplantation is prone to the risk of immune rejection, infection and disease transmission; ordinary artificial Synthetic mate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435G01N33/68C12Q1/02C12N5/0775C12N5/077A61L27/22A61L27/54
CPCC07K14/43509G01N33/68G01N33/5014C12N5/0654A61L27/227A61L27/54C12N2506/1353C12N2501/998C12N2500/42C12N2500/38C12N2501/39A61L2430/02A61L2300/252A61L2300/412C08L89/00
Inventor 宋俊祎胡碧茹李保山梁超叶宗煌曾玲刘梁程淦克胜吴吉喆唐佳文
Owner NAT UNIV OF DEFENSE TECH
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