Nucleic acid aptamer wyz-5 of ovarian mucinous carcinoma cell 3ao and its screening method and application
A nucleic acid aptamer, WYZ-5 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, etc. , to achieve good pharmacokinetic properties, no immunogenicity, and good reproducibility
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Embodiment 1
[0045] Example 1: Screening of nucleic acid aptamer WYZ-5:
[0046] The screening method of the nucleic acid aptamer WYZ-5 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-5:
[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 Sequencing 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-5 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-5 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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