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
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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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