Calcium binding peptides
A technology of calcium-binding peptides and compositions, applied in the direction of peptides, specific peptides, drug combinations, etc., can solve problems such as increasing the nucleation rate of hydroxyapatite
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Embodiment 1
[0073] Example 1: Binding of DSS peptide to calcium hydroxyapatite:
[0074] Production of Asp- Ser-Ser (DSS) repeats of four DSS peptides. Labeled with fluorescein, various concentrations of labeled peptides (0-100 μM) were quantified (0.3 mg) with a specific surface area of 100 m 2 / g Hydroxyapatite nanocrystals (Berkeley Advanced Biomaterials, Inc.) were mixed. Samples were incubated for 10 minutes, followed by centrifugation to remove hydroxyapatite. The amount of peptide in the mixture before and after removal of hydroxyapatite was determined based on the spectral absorbance at 480 nm (absorption peak of the fluorescein label). By combining the final absorption (A f ) and early (A i ) absorption and initial concentration (P 0 ) ratio for comparison [P 结合 =(A f / A j ) P 0 ] to calculate the amount of peptide binding. Generate a graph illustrating equilibrium, per m 2 The amount of peptide bound and the concentration of unbound peptide on the surface area of...
Embodiment 2
[0077] Example 2: Binding of DSS Peptides to Teeth:
[0078] To demonstrate the ability of DSS peptides to bind to biological tissue, sagittal sections of human teeth were incubated for 10 minutes in 12.5 μM 5(6) carboxyfluorescein-labeled 6DSS (SEQ ID NO: 14) peptide solution containing 10 mM NaCl and 50 mM HEPES at pH 7.0. Control samples were prepared without peptide. Treated samples were washed and imaged using a confocal laser scanning microscope (CLSM) with a blue laser (excitation wavelength (λ) = 488 nm) and a FITC emission filter. Strong fluorescent staining indicated that 6DSS (SEQ ID NO: 14) was bound to the tooth surface ( figure 1 D). Sham-treated control sections exhibited no fluorescence. Peptide binding is restricted to dentin ( figure 1 D, light color area on the left), no combination was seen in the enamel area ( figure 1 D, dark area on the right).
Embodiment 3
[0079] Example 3: Binding of DSS Peptides to Mineralized Mouse Bone Marrow Nodules:
[0080] Mouse bone marrow cultures were grown to confluence in DMEM+10% FBS, and then labeled with 2.5 μM 5(6)-carboxyfluorescein (SEQ ID NO: 14 ) or 2.5 μM 5(6)-carboxyfluorescein-labeled peptide #3-1 (missense control peptide, SEQ ID NO: 24) in aMEM+10% FBS solution was continuously treated for 3 weeks. Cultures were imaged by fluorescence microscopy with FITC excitation / emission filter sets to obtain brightfield and fluorescence images. Intense staining was observed in DSS-treated samples, as figure 2 Indicated by light color in the central nodular block in B. No staining was observed in the control samples ( figure 2 C, 2D), showing that the 6DSS (SEQ ID NO: 14) peptide specifically binds mineralized nodules in mouse bone marrow cultures.
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