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Kidney injury molecule-1 (kim-1) targeting polypeptide and application thereof

A targeted peptide and targeting technology, applied in the field of kidney injury molecule-1 targeted peptide, can solve the problems of limited kidney methods, and achieve the effects of easy operation, high reaction efficiency, and wide sources

Active Publication Date: 2022-05-17
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, methods to specifically target damaged kidneys remain limited

Method used

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  • Kidney injury molecule-1 (kim-1) targeting polypeptide and application thereof
  • Kidney injury molecule-1 (kim-1) targeting polypeptide and application thereof
  • Kidney injury molecule-1 (kim-1) targeting polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Screening and Verification of Kim-1 Targeting Polypeptides

[0034] 1. Screening of Kim-1 targeting peptides

[0035] The experimental steps are as follows:

[0036] (1) 40 μg of Kim-1 protein recombined in vitro was coated onto an ELISA plate, and coated overnight at 4°C;

[0037] (2) Wash 3 times with PBS containing 0.05% Tween to remove excess antigen;

[0038] (3) Add 5% BSA blocking solution, block at 30°C for 2 hours;

[0039] (4) Remove the blocking solution, add phage random display library (1011pfu), and incubate at 37°C for 2 hours;

[0040] (5) Wash 5 times with PBS containing 0.05% Tween to wash away unbound phage;

[0041] (6) Add Glycine-HCl buffer (pH 2.2) to elute the bound phage, then add Tris-HCl buffer (pH 9.1) to neutralize;

[0042] (7) The eluate was titered and amplified with Escherichia coli TG1;

[0043] (8) Repeatedly panning the Kim-1 protein by using the phage product recovered and amplified by the last round of panning accord...

Embodiment 2

[0059] Example 2 Construction and identification of kidney-targeted delivery system

[0060] 1. Extraction of erythrocyte-derived extracellular vesicles

[0061] (1) Collect mouse blood and add it to a sterile centrifuge tube containing anticoagulant ACD (1.32% sodium citrate, 0.48% citric acid, 1.47% glucose), and dilute the blood with an equal amount of PBS;

[0062] (2) Slowly add the diluted blood into the Ficoll-Paque (GE Healthcare) separation solution containing the same volume, and centrifuge at 500g, 4°C for 30min;

[0063] (3) Carefully collect the red blood cells at the bottom layer, wash with PBS for 3 times, and resuspend in a sterile centrifuge tube with PBS;

[0064] (4) Add 10 μmol / L calcium ionophore (Abcam), and treat for 48 hours at 4° C. on a rocking table.

[0065] (5) Extracellular vesicles were extracted by ultracentrifugation: centrifuge at 4°C, 2000g for 20 minutes to remove red blood cells; carefully transfer the supernatant to a new sterile centrif...

Embodiment 3

[0076] Example 3 Ability Test of Kidney Targeted Delivery System Targeting Damaged Kidney

[0077] Mice underwent unilateral (right) renal ischemia-reperfusion surgery, and the renal ischemia time was 35 minutes; after renal reperfusion, 200 μg of REV were injected through the tail vein LTH or REV Scrbl As a control (both the targeting polypeptide LTH and the control peptide Scrbl were labeled with FITC); mice were sacrificed 12 hours after injection, and kidneys were retained for fluorescence imaging (IVIS Spectrum imaging system, PerkinElmer) and immunofluorescence detection. see results Figure 4 , compared to the control REV Scrbl , REV LTH Accumulation in injured kidneys increased 4-fold, suggesting that REV LTH can effectively target the damaged kidney; in addition, REV LTH Can specifically target Kim-1-positive renal tubules.

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Abstract

The invention discloses a kidney injury molecule-1 (Kim-1) targeting polypeptide and an application thereof, belonging to the field of biomedicine. The Kim-1 targeting polypeptide disclosed in the present invention is preferably LTH, and its amino acid sequence is: LTHVVWL; it specifically binds to damaged kidneys. Based on the targeting polypeptide LTH, the present invention also provides a targeted delivery system that can target damaged kidneys.

Description

technical field [0001] The disclosure belongs to the field of biomedicine, and in particular relates to a kidney injury molecule-1 (Kim-1) targeting polypeptide and application thereof. Background technique [0002] Kidney injury molecule-1 (Kim-1) is a type I transmembrane glycoprotein composed of extracellular Ig-like domain, transmembrane domain and intracellular domain. In healthy kidneys, Kim-1 is hardly expressed; however, in kidney diseases caused by various reasons, Kim-1 is specifically highly expressed in damaged renal tubular epithelial cells, and has become an early stage of various kidney diseases such as acute kidney injury. diagnostic markers. At the same time, the kidney-specific expression of Kim-1 also suggests that targeting Kim-1 can be used as a powerful strategy for precise treatment of kidney diseases. Therefore, the present invention utilizes phage display technology to screen and obtain the polypeptide specifically binding to Kim-1 protein, and con...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K31/713A61K47/62A61K47/69A61P13/12
CPCC07K7/06A61K31/713A61K47/62A61K47/6901A61P13/12
Inventor 刘必成汤涛涛王彬
Owner SOUTHEAST UNIV
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