A kind of nucleic acid aptamer that specifically binds dek protein and its application
A nucleic acid aptamer and specific technology, applied in the field of rheumatoid arthritis drug research, can solve the problem of low stability of aptamers, inability to prepare drugs for preventing and treating rheumatoid arthritis, low bioavailability, etc. problems, to achieve the effects of improving stability, inhibiting neutrophil production, and being easy to prepare
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] Biofilm Interferometry Determination of Intermolecular Interactions:
[0035] The kinetics of nucleic acid aptamer binding to DEK protein was determined by the forteBIO Octet Red96 biomolecular interaction detection platform. Using the interference phenomenon of light, there is a biosensor inside the instrument, and the bottom of the sensor is covered by a biofilm. When visible light with a certain width is perpendicular to the biofilm layer, the light is reflected at the two interfaces of the biofilm layer to form an interference wave. When immobilized aptamer molecules bound to DEK protein in kinetic buffer, biofilm thickness increased, and binding kinetic data were calculated based on the phase shift of light waves.
[0036] The aptamers used in the experiment were all biotin-modified, and the purchased biotinylated aptamer powder was diluted to 100nM with kinetic buffer, and the DEK recombinant protein was entrusted to Wuhan Huamei Bioengineering Co., Ltd. The pur...
Embodiment 2
[0038] Denaturing urea polyacrylamide gel electrophoresis to determine the stability of the nucleic acid aptamer DTA1-DTA1.4 in vitro cell culture conditions within 6 hours:
[0039] Dilute the nucleic acid aptamer to 2.5 μM with RPMI1640 medium containing 2% BSA, incubate at 37°C for 0, 1, 2, 4, 4.5, and 6 hours, and add 3× formamide gel immediately after reaching the time The loading buffer and the sample were mixed thoroughly at a ratio of 1:2 and stored at -20°C.
[0040] Assemble the gel glass plate, check for leaks for 10 minutes, and prepare 12% denatured urea-polyacrylamide gel: add 4 grams of urea, 4 mL of 30% acrylamide, and 2 mL of 5×TBE to the beaker, and place in a water bath at 50 °C dissolved in. After dissolving, cool to room temperature, add 0.1 mL of 10% ammonium persulfate and 4 μL of TEMED, mix well, inject the glue between the glass plates, insert a comb, and polymerize for 30 min. Install the electrophoresis device, fill the electrophoresis tank with 1×...
Embodiment 3
[0042] Denaturing urea polyacrylamide gel electrophoresis to determine the stability of the nucleic acid aptamer DTA1-DTA1.4 in 90% mouse serum within 24 hours:
[0043] The nucleic acid aptamer was mixed with the mouse serum so that the final concentration of the aptamer was 2.5 μM and the final concentration of the serum was 90%. Incubate at 37°C for 0, 2, 4, 6, 8, 12, and 24 hours respectively, and immediately add an equal volume of phenol:chloroform:isoamyl alcohol P:C:I (25:24:1) after the time is reached, After sufficient shaking, centrifuge at 10,000 RPM for 10 minutes at 4°C, take out the supernatant, fully mix the supernatant with 3× formamide gel loading buffer at a ratio of 2:1, and store at -20°C. Denaturing urea polyacrylamide gel electrophoresis process is as described in Example 2. Electropherogram as Figure 5 As shown, DTA1, DTA1.1, DTA1.2, and DTA1.3 were almost completely degraded within 24 hours, while DTA1.4 was not significantly degraded under this cond...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More