Polypeptide specifically binding to CD34 molecule and application thereof
A specific and molecular technology, applied in the field of biomedicine, can solve the problems of legacy medical safety hazards, large preparation workload, long production cycle, etc., and achieve the effect of reducing stem cell transplantation complications, good permeability and low cost.
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Embodiment 1CD34
[0049] Example 1 The screening of CD34-binding phage
[0050] 1) Enrichment of CD34-binding phages with CD34 as the target molecule
[0051] The 96-well plate was coated with 5 μg / mL CD34 molecule (purchased from PeproTech), and the T7 phage lysate displaying random polypeptides in the T7 human cDNA library (Novagen) was mixed with 3% skimmed milk powder at a mass ratio of 1:1. , and incubated with CD34 molecule-coated 96-well plate for 30 minutes. Then, the non-specifically bound phages were eluted with washing solution, and the T7 phages bound to CD34 were enriched. After repeated screening, the recovery rate of the phage library was no longer increased. Petri dishes not coated with CD34 molecules were used as the control group.
[0052] The titer of phage input in the first round of screening was 6.4×10 10 cfu, the recovered phage titer was 5.4×10 5 cfu, the recovered phage was amplified, and the titer reached 4.2×10 9 cfu, continue to invest in the second round of scr...
Embodiment 2CD34
[0060] Example 2Co-immunoprecipitation of CD34 binding polypeptide
[0061] (1) Total protein extraction: Wash K562 cells once with pre-cooled PBS buffer, discard PBS, add 500 μL of non-denatured protein lysate containing protease inhibitors to the culture dish, and collect the cells into a 1.5 mL centrifuge tube , sonicated 4 times, 4 seconds each time, placed on ice for 30 minutes, centrifuged at 8000 rpm at 4°C for 20 minutes, and transferred the supernatant to a new centrifuge tube to obtain the total cell protein product;
[0062] (2) Pretreatment of total cell protein product: add 50 μL normal goat serum blocking solution to the total cell protein product, incubate on ice for 1 hour, then add 100 μL protein G microsphere suspension (Calbiochem), and incubate at 4°C for 20 minutes , centrifuging to remove protein G microspheres;
[0063] (3) Co-immunoprecipitation: Add CD34 antibody (AR) to the total protein pretreated in step (2) as the experimental group, and add the s...
Embodiment 3
[0068] Example 3 Binding of CD34-binding polypeptides to CD34 molecules of CD34-positive cells
[0069] (1) Cell preparation: place K562 cell suspension at 37°C, 5% CO 2 cultured in an incubator. When the cell density reaches 80%, centrifuge to discard the culture medium, wash once with PBS buffer, centrifuge, take the cell suspension, drop it on the glass slide coated with poly-lysine, fix it with cold acetone after drying for 10 minute.
[0070] (2) Wash with PBS buffer 3 times, 5 minutes each time. Then block with 5% BSA blocking solution for 1 hour at room temperature.
[0071] (3) Primary antibody incubation: the primary antibody dilution of CD34 antibody (AR) was dropped onto the glass slide, and incubated overnight at 4°C. Wash with PBS buffer 3 times, 5 minutes each time.
[0072] (4) Secondary antibody incubation and peptide binding: Peptides No. 17 and No. 19 were added to the diluted AlexaFluor561-labeled anti-rabbit antibody and mixed, then dropped onto the co...
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