Nucleic acid aptamer WYZ-4 of ovarian mucinous cancer cells 3AO and screening method and application thereof
A nucleic acid aptamer and screening method technology, applied in the screening process, pharmaceutical formulations, biochemical equipment and methods, etc., can solve the problems of lack of specific markers of ovarian mucinous carcinoma, unsatisfactory pertinence, instability and the like, Achieve good pharmacokinetic properties, no immunogenicity, and good reproducibility
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Embodiment 1
[0045] Example 1: Screening of nucleic acid aptamer WYZ-4:
[0046] The screening method of the nucleic acid aptamer WYZ-4 of the ovarian mucinous carcinoma cell 3AO comprises the following steps:
[0047] (1) Preparation of screening library:
[0048]Entrust Sangon Bioengineering Co., Ltd. to synthesize a single-stranded DNA library (5'-AGCTGCTATACTACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTAGCGATAGCTAGCTT-3'); take 1 tube of 1OD single-stranded DNA library, centrifuge at high speed for 2min; gently open the tube cap in a fume hood, add 300μL binding Buffer (4.5g / L glucose in DPBS, 100mg / L yeast tRNA, 1g / L BSA, 5mM MgCl 2 ), vortex and oscillate to dissolve; seal the cap of the centrifuge tube with parafilm, bathe in 95°C water for 5 minutes, rapidly ice-water bath for 2 minutes; centrifuge at high speed for 2 minutes, and set aside.
[0049] (2) Reverse screening of SKOV3 cells: inhale the library prepared in step (1) into a 100mm-diameter petri dish with a gro...
Embodiment 2
[0057] Example 2: Analysis of the sequence of the nucleic acid aptamer WYZ-4:
[0058] After 12 rounds of screening, the positive screen supernatant of 3AO cells was recovered, and Shanghai Passino Biotechnology Co., Ltd. was entrusted to use high-throughput sequencing technology to analyze the library sequence. The analysis process is: PCR amplification enrichment library, and adding Sequence adapters and Index parts were placed; the purified library was selected by gel electrophoresis; the quality control of the library was performed using the Agilent 2100 Bioanalyzer through the Agilent High Sensitivity DNA Kit; the library was quantified using the Quant-iT PicogGreen dsDNAAssay Kit; Bridge PCR amplification of the template, annealing of sequencing primers, and sequencing while synthesizing; and comparison and enrichment analysis of the sequencing results.
[0059] Using the UNAFold network platform to analyze under physiological conditions (37°C, 0.137M Na + , 0.001M Mg ...
Embodiment 3
[0060] Example 3: Analysis of the binding ability of nucleic acid aptamer WYZ-4 to ovarian mucinous carcinoma cell 3AO:
[0061] 1. Inoculate the target cells 3AO in a 24-well plate (50,000 / well), at 37°C, 5% CO 2 Cultivate for 24h.
[0062] 2. Configure the FITC-labeled single-stranded DNA (including the nucleic acid aptamer WYZ-4 and the unscreened single-stranded DNA library) into a solution with a concentration of 0nM, 10nM, 50nM, 100nM, and 200nM with a binding buffer; 95°C Water bath for 5 minutes, rapid ice water bath for 2 minutes.
[0063] 3. Wash the cells twice with binding buffer, add 200 μL of the single-stranded DNA solution with the above-mentioned gradient concentration to each well, and incubate on ice for 30 minutes.
[0064] 4. After incubation, discard the supernatant and scrape the cells with a cell scraper; wash twice with binding buffer, and resuspend the cells with a volume of 250 μL.
[0065] 5. Use the FACSAria flow cytometer of BD Company to measu...
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