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Preparation and application of polypeptide for promoting bone regeneration or bone formation

A bone regeneration and multipurpose technology, applied in the field of biomedicine, can solve the problems of increasing the risk of cancer development, poor protein stability, and hindering clinical application, etc., and achieve the effect of simple structure, small molecular weight, and stable structure

Pending Publication Date: 2022-08-05
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fast release kinetics and poor protein stability of BMPs require supraphysiological doses to maintain a sufficiently high local concentration to play a role, which hinders its clinical application due to potential adverse effects, such as postoperative edema, heterotopic bone, etc. formation, bone graft resorption, osteolysis, and increased risk of cancer development, etc.

Method used

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  • Preparation and application of polypeptide for promoting bone regeneration or bone formation
  • Preparation and application of polypeptide for promoting bone regeneration or bone formation
  • Preparation and application of polypeptide for promoting bone regeneration or bone formation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The present embodiment provides a method for preparing a polypeptide that promotes the osteogenic function of BMP2, comprising the following steps:

[0061] 1. Construction of Overexpression Plasmid

[0062] 1.1 Obtaining primers

[0063] Using pGEX-4T-1 as a vector, BamH I and Not I were selected as restriction sites, and primers for different LRR fragments in OMD were designed: OMD_LRRNT, OMD_LRR1-3, OMD_LRR4-6, OMD_LRR7-9, OMD_LRR10-11.

[0064] The amino acid sequence (SEQ ID NO:2) of OMD_LRRNT is: VPFHQYTL GCVSECFCPT NFPSSMYCDNRKLKTIPNIP M;

[0065] The amino acid sequence (SEQ ID NO:3) of OMD_LRR1-3 is: HIQQLYLQF NEIEAVTANS FINATHLKEINLSHNKIKSQ KIDYGVFAKL PNLLQLHLEH NNLEEFPFPL PKSL;

[0066] The amino acid sequence (SEQ ID NO: 4) of OMD_LRR4-6 is: ERLLLG YNEISKLQTN AMDGLVNLTMLDLCYNYLHD SLLKDKIFAK MEKLMQLNLC SNRLESMPPG LPS;

[0067] The amino acid sequence (SEQ ID NO: 5) of OMD_LRR7-9 is: SLMYLSL ENNSISSIPE KYFDKLPKLHTLRMSHNKLQ DIPYNIFNLP NIVELSVGHN KLKQ;

[00...

Embodiment 2

[0114] Embodiment 2 Alkaline phosphatase activity detection

[0115] Alkaline phosphatase (ALP) is a marker enzyme of mature osteoblasts and one of the markers of osteoblast differentiation. It can increase the concentration of local inorganic phosphoric acid and participate in the formation, metabolism and regeneration of calcified tissue. pNPP is a commonly used chromogenic substrate for phosphatase. Under alkaline conditions, it can generate para-nitrophenol under the action of alkaline phosphatase, which is a yellow product. The absorbance can be detected at 400-415nm. The darker the yellow product, the higher the alkaline phosphatase detection activity.

[0116] The steps of alkaline phosphatase (ALP) activity detection in this embodiment are:

[0117] (1) Inoculate hDPSCs into a 12-well plate, and add the following protein stimulation when the cells grow to 90% density. The groups are divided into: Ctrl group (0.1% BSA solution), 0.1B group (0.1 μg / mL BMP2 in 0.1% BSA...

Embodiment 3

[0123] Example 3 OMD_LRR10-11 thermal denaturation assay

[0124] The protein unfolding transition temperature, Tm, refers to the temperature at which 50% of the protein unfolds. Proteins with high Tm are more stable because greater energy input is required to reach the transition temperature. Tm is an accurate and well-recognized measure of protein stability, so it is a parameter that must be determined. This example was used to measure the protein stability of the polypeptide OMD_LRR10-11.

[0125] The instrument used in this experiment was Prometheus (PR) (nanotemper, Germany).

[0126] The experimental steps are as follows: suck the polypeptide solution (without bubbles) obtained by the method of Example 1 with a certain concentration with a capillary glass tube, and then use Prometheus (PR) to measure Tm. The folded state of the protein is observed by detecting the change of the endogenous fluorescence of the protein. The fluorescence intensity of a single wavelength ...

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Abstract

The invention discloses preparation and application of a polypeptide for promoting bone regeneration or bone formation. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. According to the invention, the tenth to eleventh segments of LRRs motifs are screened from eleventh segments of LRRs motifs of an OMD core protein and are key sites of a complex formed by OMD, BMP2 and a receptor thereof. The polypeptide segment can activate a downstream BMP / SMAD signal channel, reduce the dosage of BMP2 and optimize BMP2 osteogenesis. The polypeptide is small in molecular weight, simple and stable in structure and higher in activity, and can be better applied to promotion of bone injury regeneration and repair.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to the preparation and use of a polypeptide for promoting bone regeneration or bone formation. Background technique [0002] Oral and maxillofacial bone defects caused by congenital diseases, cancer, trauma, infection, etc. seriously affect the quality of life of patients. Tissue engineering, the combination of stem cells, scaffolds and growth factors, offers new hope for functional bone regeneration. A variety of growth factors are involved in regulating different stages of the bone regeneration process; some of these growth factors have been widely used in preclinical models of bone defect repair. Mainly include: Bone Morphogenetic Proteins (BMPs), vascular endothelial growth factor-A, platelet-derived growth factor-BB, fibroblast growth factor-2, etc. Among them, bone morphogenetic protein 2 (BMP2) is the most widely used growth factor for bone transplantation adjuv...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/70C12N15/66C12P21/02A61K38/17A61K38/18A61P19/08
CPCC07K14/4702C12N15/70A61K38/1709A61K38/1875A61P19/08A61K2300/00Y02A50/30
Inventor 黄正蔚朱晓晗林文珍陈燕
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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