Nucleic acid aptamer sequences capable of specifically recognizing deta-glucuronidase and application of nucleic acid aptamer sequences capable of specifically recognizing deta-glucuronidase
A technology of aldolidase nucleic acid and nucleic acid aptamer, which is applied in the direction of hydrolytic enzymes, DNA/RNA fragments immobilized on or in inorganic carriers, etc., and can solve the problem of nucleic acid aptamers with high specificity and high affinity Preparation methods and applications
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Embodiment 1
[0014] Embodiment 1: the preparation of β-glucuronidase PGUS-E
[0015] Inoculate the strain containing the plasmid pET-28a-GUS / BL21(DE3) into 30ml of LB (Kanamycin 50μg / ml), prepare the seed solution overnight at 37°C, and transfer it to 500ml of fresh LB (Kanamycin 50 μg / ml) medium, cultured at 37°C until OD 600 =~0.6, add isopropyl-1-thio-β-D-galactopyranoside (0.5 mM) and induce at 16°C for 5 hours. After induction, the cells were centrifuged at 12,000 rpm at 4°C for 10 minutes, the supernatant was discarded to collect the cells, the cells were resuspended in protein breaking buffer for washing, and centrifuged at 12,000 rpm at 4°C for 10 minutes. Finally, the bacteria were resuspended in 20ml of protein buffer, and ultrasonically disrupted in an ice bath. The sonicated bacterial solution was centrifuged at 4° C. at 12,000 rpm for 20 minutes, and the supernatant was collected to obtain a crude enzyme solution.
[0016] Purification of β-glucuronidase PGUS-E. The crude ...
Embodiment 2
[0017] Embodiment 2: Preparation and identification of the nucleic acid aptamer of β-glucuronidase PGUS-E
[0018] The aptamers of PGUS-E were selected by magnetic separation using β-glucuronidase PGUS-E immobilized on Ni-NTA-magnetic beads. The selected primary nucleotide random library ssDNA (Apt-selection) was chemically synthesized by Beijing Jinweizhi Biological Company and purified by HPLC. (Apt-selection: 5'-AGCAGCACAGAGGTCAGATGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA-3').
[0019] Nucleic acid aptamer enrichment so primers:
[0020] Apt-F: 5'-AGCAGCACAGAGGTCAGATG-3', chemically synthesized (HPLC purification).
[0021] Apt-R: 5'-TTCACGGTAGCACGCATAGG-3', chemically synthesized in phosphorylated and unphosphorylated forms, respectively (HPLC purification)
[0022] 10 μM nucleotide random library DNA was incubated with PGUS-E immobilized on Ni-NTA-magnetic beads for 30 minutes at room temperature. Aptamers bound and unbound to PGUS-E were separated mag...
Embodiment 3
[0028] Example 3: Purification and immobilization application of PGUS-E nucleic acid aptamer to PGUS-E
[0029] For the purification assay of PGUS-E nucleic acid aptamers to PGUS-E, incubate biotinylated nucleotide aptamers with streptomycin-modified magnetic beads at room temperature for 10 minutes, heat to 95°C for 5 minutes, and Quickly chill on ice for 3 minutes. Incubate the magnetic beads immobilized with nucleic acid aptamers and the PGUS-E crude enzyme solution on a shaker at room temperature for 30 min, separate the magnetic beads on a magnetic stand to discard the liquid, and wash the magnetic beads 3 times with washing buffer. The purity and quantity of the purified β-glucuronidase PGUS-E were detected by SDS-PAGE electrophoresis and BCA method, respectively. The separation efficiencies of the nucleic acid aptamers Apt5 and Apt9 to isolate and purify PGUS-E protein were 31.75 μg / mg and 32.95 μg / mg, respectively. At the same time, the immobilization efficiency of β...
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