Aptamer in specific binding with perforin and application of aptamer
A nucleic acid aptamer and specific binding technology, applied in the field of aptamers
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Embodiment 1
[0031] Example 1, carboxyl magnetic beads coated perforin protein
[0032] Take 1ml of dimethyl fumarate (dimethyl formaide) dissolved containing 2 × 10 9 / ml carboxyl magnetic beads to EP tube, put on the magnetic stand for 4 minutes, carefully discard the supernatant, add 2ml of 0.1mol / L sodium phosphate (pH7.4), mix for 30 seconds, let stand for 10 minutes; put on the magnetic stand 2 Minutes, carefully remove the supernatant; then resuspend the magnetic beads with 2ml of 0.1mol / L sodium phosphate; place on the magnetic stand for 2 minutes, carefully remove the supernatant, add 600μl of 0.1mol / L sodium phosphate, add 600μl perforin protein Add 600 μl of ammonium sulfate with a concentration of 3 mol / L to the solution with a concentration of 1 mg / ml, and mix well; incubate at 37°C for 20 hours, shake slowly, then place on the magnetic stand for 4 minutes, and carefully invert the magnetic stand to ensure that all the magnetic beads are collected. Aspirate the supernatant an...
Embodiment 2
[0034] Example 2, SELEX screening of perforin-specific binding oligonucleotide aptamers
[0035] Construct a random ssDNA library (synthesized by Beijing Quanshijin Biotechnology Co., Ltd.) with a length of 81 nt. The specific sequence is as follows: 5'-cccctgcaggtgattttgctcaagt-(N35)-agtatcgctaatcaggcggat-3', with fixed sequences at both ends and 35N in the middle Represents 35 random nucleotides with a library capacity of approximately 10 10 , the sequence of the library is the P81ss library.
[0036] The random oligomeric ssDNA library was then amplified with the following primers:
[0037] P81-SP:5'-cccctgcaggtgattttgctcaagt-3' (SEQ ID NO.1);
[0038] P81-AP: 5'-Biotin-atccgcctgattagcgatact-3' (SEQ ID NO.2);
[0039] Random ssDNA library (first round screening volume is 2000pmol, equivalent to 2 OD random libraries) was dissolved in EP tube (coated with 1% BSA, blocked overnight at 4°C) with 1ml 1× selection buffer, denatured at 95°C for 5min; after denaturation Immedi...
Embodiment 3
[0051] Example 3. Detection of perforin aptamers by cyclic voltammetry
[0052] The 5' ends of the screened six aptamers were modified with sulfhydryl groups to form 5'-SH-(CH2)6-aptamer sequence-3', and then the sulfhydryl groups bonded the aptamers to the gold-plated aptamers through Au-S bonds. The surface of the glassy carbon electrode was then blocked with hexanethiol to obtain a perforin aptamer-modified electrode. The specific steps are as follows: Wet the surface of the gold-plated glassy carbon electrode, and then sequentially add 0.3 μm and 0.05 μm Al 2 o 3 The powdered suede was used to polish and polish the electrode. After each grinding, the electrode was washed with ultrapure water, and then ultrasonically washed in ethanol and ultrapure water for 5 minutes. The treated gold electrode was dried for later use; Add 20 μL of 2 μM 5’ end thiol-modified solution to the surface dropwise, modify at room temperature overnight, wash the electrode with ultrapure water, t...
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