Targeted ADGB inhibitory RNA and application thereof in preparing antitumor drugs
A DNA sequence and drug technology, applied in anti-tumor drugs, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of difficult to locate nerve damage, poor efficacy of anti-tumor drugs, etc.
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Embodiment 1
[0040] Example 1: Design of siRNA and preparation of interference carrier
[0041] The complementary DNA (Complementary DNA, cDNA) sequence information of ADGB mRNA (Genebank Accession: NM_024694.3) was downloaded from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ). Using BLOCK-iT from Life Technologies TM RNAi Designer designed shRNA targeting ADGB and obtained 10 target sequences.
[0042]In NCBI's homologous sequence comparison analysis nucleotide blast, the target sequence is input for comparison analysis. It is required that the target sequence has no high homology with other human mRNA genes, and can be used as an interference target that specifically interferes with ADGB. According to these sequences, artificially design shRNA in the order of Sense-loop-Antisense, sense refers to the sense strand of the target sequence, antisense refers to the antisense strand of the target sequence, and loop refers to the sequence forming a loop, and UUCAAGAGA is used here. Then add...
Embodiment 2
[0052] Example 2: Interfering with the packaging of lentiviral particles
[0053] At 37°C 5% CO 2 HEK293T cells (hereinafter referred to as 293T, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured in a cell incubator. The medium used was DMEM (Gibco, New York, USA) supplemented with 10% fetal bovine serum (Gibco, New York, USA). production) medium. Subculture 293T cells in a petri dish with a diameter of 10 cm. When the cells grow to a confluence of about 50%, culture them in serum-free medium for 4 hours. According to the instructions of Lipofectamine2000 (produced by Invitrogen, USA), prepare 22.5 μg of the pLKD-CMV-GFP-U6-shRNA plasmid obtained above (or empty vector plasmid without shRNA), 7.9 μg of the virus coat plasmid psPAX2 (Nuen, Shanghai, China) Biotechnology Co., Ltd.), 14.6 μg of the transfection mixture of the packaging plasmid pMD2.G (provided by Shanghai Nuen Biotechnology Co., Ltd., China)....
Embodiment 3
[0056] Example 3: Verification of interference efficiency at the mRNA level
[0057] Glioma cell line U373 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) was cultured at 37°C in 5% CO 2 In the cell culture incubator, the medium was DMEM medium supplemented with 10% fetal bovine serum. The cells were cultured in a 6cm-diameter dish to approximately 30% confluence, and the control lentivirus infection particles and the virus infection particles interfering with ADGB were added respectively (the multiplicity of infection was 10), and the cells were continued to be cultured for 4 days. Rinse the cells twice with PBS, each time with 3ml of PBS, then digest the cells with 2ml of 0.25% (w / v) trypsin, and collect the cells by centrifugation at 1000 rpm. Afterwards, the total RNA was extracted with the reagent Trizol (produced by Invitrogen, USA), and the operation was performed according to the instructions. Use the RT M-ML...
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