A nucleic acid aptamer specifically binding to Sudan Red and its application
A technology of nucleic acid aptamer and Sudan red, applied in the field of biomedicine
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Embodiment 1
[0024] Example 1: Screening of Sudan Red Nucleic Aptamer (C07)
[0025] 1. Coupling of Ligand (Sudan Red) and Matrix (Sepharose 6B)
[0026] 1. Suspend 1 g of lyophilized powder Sepharose 6B in 3 mL of distilled water (1 g of lyophilized powder can yield a final matrix volume of approximately 3.0 mL);
[0027] 2. Immediately rinse with distilled water at about 200 mL per gram of powder for 1 hour in a sintered glass filter (porosity G3);
[0028] 3. Dissolve 6 g of ligand with 6 mL of coupling buffer solution (0.05 M, carbonate buffer solution at pH 9.6) to make the final concentration 1 mg / mL, or transfer the dissolved ligand with a desalting column In the coupling buffer solution, adjust the pH value of the aqueous phase;
[0029] 4. Adjust the ratio of the matrix to the buffer solution with a concentration of 1 mg / mL dissolved in the ligand in step 3 to a volume ratio of 1:2, mix for 16 h in a water bath at 25°C to 40°C, and 37 ℃ shaker overnight;
[0030] 5. At 40°C to...
Embodiment 2
[0076] Example 2: Cloning and sequencing of nucleic acid aptamers and prediction of single-stranded DNA secondary structure
[0077] 1. Preparation of Escherichia coli DH5α Competent Cells
[0078] 1. Pick a single DH5α colony, inoculate it in 3 mL of LB medium without ampicillin, and incubate overnight at 37°C. The next day, take the above bacterial solution and inoculate it in 50 mL of liquid LB medium at a ratio of 1:100. Shake at ℃ for 2 hours; when the OD600 value reaches 0.35, harvest the bacterial culture;
[0079] 2. Transfer the bacterial culture to a 50 mL pre-cooled sterile polypropylene tube and place it on ice for 10 min to cool the culture;
[0080] 3. Centrifuge at 4000 rpm for 10 min at 4°C, discard the culture medium, and invert the tube for 1 min to drain the remaining culture medium;
[0081] 4. Add 150 μL ice-cooled 0.1 mM CaCl each 2 Solution, combine two tubes, ice bath for 10 min;
[0082] 5. Centrifuge at 4000 rpm for 10 min at 4°C, discard the supe...
Embodiment 3
[0096] Embodiment 3: circular dichroism to K + Research on the relationship between concentration and nucleic acid aptamer C07
[0097] 1. After diluting the aptamer to 20 μM with 20 mM Tris-HCl buffer (pH 7.2), denature at 94°C for 0.5 min and cool to 25°C at a rate of 0.5°C / min.
[0098] 2. Dilute the aptamer C07 to 2.5 μM with 20 mM Tris-HCl buffer (pH7.2) containing different concentrations (0, 5, 10, 20, 50 mM) of KCl.
[0099] 3. Detect with a circular dichroism spectrometer at 25 °C at a wavelength of 220–340 nm (results in figure 1 ), the results showed that the nucleic acid aptamer C07 had a peak at 275 nm in different solutions, indicating that the nucleic acid aptamer had a stem-loop and G-quadruplex structure.
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