Oligonucleotide aptamer specifically binding to lung cancer serum and application
An oligonucleotide and aptamer technology, applied in the field of molecular biomedicine, can solve the problem of lack of lung cancer tumor markers, and achieve the effect of broad clinical application prospects and basic application prospects.
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Embodiment 1
[0035] Preparation of oligonucleotide aptamer Cell APT-2
[0036] (1) Construction of initial oligonucleotide library (SEQ ID NO.2):
[0037]5'-CTATAGCAATGGTACGGTACTTCC-(40N)-CAAAAGTGCACGCTACTTTGCTAA-3'
[0038] Wherein, N represents any base among A, T, C, and G, and the fixed sequences at both ends of the oligonucleotide library are PCR amplification primer binding regions, and the middle is a random sequence of 40 bases.
[0039] (2) Treatment of cell culture medium
[0040] All cell lines come from the ATCC cell bank. Lung cancer cells A549 and lung epithelial cells HMVEC were cultured in high-glucose DMEM medium. When the growth reached about 80%, they were replaced with phenol red-free and serum-free DMEM medium for 24 hours. to collect. Collect the collected cell culture medium into a 1.5mL centrifuge tube, centrifuge at 1000g, 4°C for 5min, remove suspended cells and dead cells, collect the supernatant after centrifugation, continue to centrifuge at 2000g, 4°C for 5...
Embodiment 2
[0059] Detection of binding ability of oligonucleotide aptamer Cell APT-2 to culture medium of lung cancer cells by q-PCR
[0060] Prepare equal volumes of oligonucleotide aptamer Cell APT-2 with different concentration gradients (0nM, 12.5nM, 25nM, 50nM, 100nM, 200nM and 400nM), and rotate with the lung cancer cell culture medium at room temperature according to the method described in Example 1 Incubate for 1 h, then incubate with activated magnetic beads at 37°C for 1 h to form a complex. Wash once with 1 mL of screening buffer containing 0.1% Tween-20 to remove oligonucleotide aptamers that non-specifically bind to non-target proteins, denature at 95°C, collect supernatant, and perform the same q as in Example 1 -PCR analysis, the relative fluorescence intensity RFU (1 / Ct × 10 3 ) as a monitoring indicator, the results are as follows figure 2 As shown, the equilibrium dissociation constant (Kd) of the obtained oligonucleotide aptamer Cell APT-2 is 9.026±3.244nM.
Embodiment 3
[0062] Verification of specific recognition of oligonucleotide aptamer Cell APT-2 and lung cancer cell culture medium by q-PCR
[0063] The aptamer Cell APT-2 was diluted to 100nM, and incubated with lung cancer cells A549 and NCI-H441, colorectal cancer cells HT-29 and SW480, cervical cancer cells HeLa, and lung epithelial cells HMVEC at room temperature. 1h, followed by incubation with magnetic beads for 1h at 37°C with rotation. Wash once with 1 mL of screening buffer containing 0.1% Tween-20 to remove oligonucleotide aptamers that bind non-specifically to non-target proteins, denature at 95°C, collect supernatant, and monitor by q-PCR. RFU (1 / Ct×10 3 ) as a monitoring indicator. The result is as image 3 As shown, the oligonucleotide aptamer Cell APT-2 has a strong binding ability to lung cancer cell lines A549 and NCI-H441, so the aptamer Cell APT-2 has a strong specific binding ability to lung cancer cell culture medium .
[0064] Table 1 Binding ability of oligonuc...
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