Leptin receptor affinity peptide and application thereof

An affinity peptide and receptor technology, applied in the field of biomedicine, can solve the problems of inability to exert differentiation and repair, achieve good tissue damage repair effect, high affinity, and improve the effect of recruitment

Active Publication Date: 2021-08-13
DUBU BIOSCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the fluidity of body fluids, MSCs that are simply dripped onto the material from an external source and circulating

Method used

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  • Leptin receptor affinity peptide and application thereof
  • Leptin receptor affinity peptide and application thereof
  • Leptin receptor affinity peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This example uses phage display technology to screen for affinity polypeptides that specifically bind to the Leptin receptor

[0057] Phage random heptapeptide display library was purchased from NEB Company, 100 µl, titer 1×10 13 pfu / ml. Stored in TBS buffer (50mM Tris-HCl, 150mM NaCl[PH7.5]) containing 50% glycerol, the storage capacity is 1.28×10 9 a transformant. Escherichia coil ER2738 is the host strain of this peptide library.

[0058] target protein coating

[0059] Dilute the Leptin receptor recombinant protein with coating buffer (0.1M sodium bicarbonate buffer [pH value 8.6]) to a final concentration of 100µg / ml, add 100µl to one well of the microtiter plate to ensure that the plate is wet, Incubate overnight at 4°C in a humid chamber.

[0060] affinity screening

[0061] The next day, pour off the coating solution in the ELISA plate, pat off the residual liquid on a clean absorbent paper, and add 200 µl of blocking solution (0.1M NaHCO 3 , 5mg / ml BSA [...

Embodiment 2

[0072] Example 2 HY7 can specifically bind to human mesenchymal stem cells

[0073] 1. Detection of the binding ability of HY7 phage monoclonal to human mesenchymal stem cells

[0074] Refer to the method in Example 1 to amplify HY7 phage monoclonal and M13KE negative control phage, and titrate. HY7 phage monoclonal and M13KE negative control phage were added to human mesenchymal stem cell well plates pre-cultured to about 80% confluence, and an irrelevant human bone marrow stromal cell line HS-5 was set as a control. Bind and elute according to the phage screening steps to obtain the HY7 phage eluate bound to the target protein and the M13KE negative control phage eluate respectively, and titrate the two according to the phage titration method to compare the binding of the two to the target molecule. The results show that HY7 can specifically bind to the surface of MSC cells, but not to the control cell HS-5, Figure 2A with Figure 2B It is a schematic diagram of the bind...

Embodiment 3

[0079] Example 3 CBD-affinity peptides can be bound to collagen materials for lung injury repair

[0080] 1. CBD-affinity peptide can be bound to collagen material

[0081] In order to avoid that the affinity peptide cannot effectively reside on the biological material and maintain an effective concentration caused by the simple adsorption method, which will affect the function of the affinity peptide. In the present invention, while synthesizing the affinity polypeptide, a short peptide CBD (the sequence is specifically TKKTLRT) with collagen-specific binding ability is introduced, synthesized at the N-terminus of the affinity polypeptide, and connected with the affinity polypeptide by a linker, the sequence is specific for GGG-K-FITC. In order to evaluate whether the CBD-affinity peptide can effectively bind to the collagen material, FITC-labeled CBD-HY7 was synthesized at the same time, and for the convenience of comparison, FITC-labeled CBD misconstructed control (sCBD-HY...

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Abstract

The invention discloses an Leptin receptor affinity peptide and an application thereof. The sequence of the Leptin receptor affinity peptide is as shown in SEQ ID No.1. The invention further discloses an affinity peptide with collagen binding capacity. The affinity peptide comprises the Leptin receptor affinity peptide and a short peptide with specific collagen binding capacity, and the short peptide with the specific collagen binding capacity is bound to the N end of the Leptin receptor affinity peptide. The affinity peptide has high affinity with a Leptin receptor and can be specifically bound with MSC, the affinity peptide is connected to a biological material, and the affinity peptide connected with the short peptide with the specific collagen binding capacity can achieve specific binding with a collagen scaffold, so that the recruitment of the biological material on the MSC with the surface expressing the Leptin receptor is improved, a better tissue damage repair effect is obtained, and an experimental basis is provided for researching the MSC in the aspects of tissue engineering repair and targeted therapy research.

Description

technical field [0001] The invention relates to a Leptin receptor affinity peptide, in particular to a Leptin receptor affinity peptide capable of specifically binding to mesenchymal stem cells (MSCs) and a screening method thereof, which is applied to tissue engineering repair and belongs to biomedical technology field. Background technique [0002] Mesenchymal stem cells (MSCs), mainly derived from mesoderm, are a type of adult stem cells with high self-renewal ability and differentiation potential, and are important members of the stem cell family. MSCs were first discovered in the bone marrow, and later studies found that MSCs mainly exist in the connective tissue of the whole body and the interstitium of organs. At present, MSCs can be isolated and prepared from bone marrow, placenta, umbilical cord, fat and other tissues. Based on the multi-differentiation potential of MSC, it can be directed to differentiate into bone, cartilage, fat, cardiac muscle and other tissue ...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61L27/22A61L27/24
CPCC07K7/06A61L27/227A61L27/24A61L2300/412C08L89/00
Inventor 戴建武庄燕
Owner DUBU BIOSCIENCES
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