siRNA module of targeted Survivin gene and application thereof
A molecular and base technology, applied in the field of siRNA molecules, can solve problems such as no expression
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[0024] Various methods can be used to prepare siRNA, such as: chemical synthesis, in vitro transcription, enzyme-cut long-chain dsRNA, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc. The emergence of these methods provides researchers with options. Better access to gene silencing efficiency.
[0025] The siRNA molecules of the present invention can be used as active ingredients of antitumor drugs. Tumor diseases include liver cancer, lung cancer, leukemia, gastric cancer, cervical cancer, multiple myeloma, skin squamous cell carcinoma, colon cancer, melanoma, bladder cancer, osteosarcoma. The inhibitory effect of the medicine of the invention on liver cancer is particularly obvious.
[0026] As another expression form of the siRNA molecule, it can be prepared in the form of a DNA expression cassette, such as: U6 promoter-siRNA transcription template-H1 promoter.
[0027] For the sake of brevity, "U6 promoter-siRNA transcription template-H1 promot...
Embodiment 1
[0039] Preparation of siRNA molecular library
[0040] 1. Main instruments, reagents and materials
[0041] 1.1 Instruments: PCR instrument (ABI company); electrotransfer instrument (BIO-RAD, MicroPulser); centrifuge (Eppendorf), long-wavelength ultraviolet lamp, etc.
[0042] 1.2 Materials and reagents: 1kb plus DNA Ladder (invitrogen); DNase I (Roche); MnCl 2 (BBI); Phosphate linker (Sigma-aldrich); ATP (BBI); BSA (NEB); BmsbI (NEB); T4 DNA ligase (NEB); Agarose (BBI); dNTP (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); Phenol Chloroform Extraction Reagent (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); Low Molecular Weight DNA Ladder (NEB); EcoP15I (NEB); T4 DNA polymerase (NEB); FokI enzyme (NEB); SfiI enzyme (NEB); Competent cells (invitrogen); pU6H1-GFP expression vector (NT Oimcs, USA). Gel extraction kit: QIAEX II Gel Extraction Kit (QIAGEN); Plasmid extraction kit (Biometics Biotechnology Co., Ltd.). Other biochemical reagents were p...
Embodiment 2
[0056] Preparation of siRNA expression cassette and screening of target sites
[0057] 1. Main instruments, reagents and materials
[0058] 1.1 Instruments: PCR instrument (ABI); real-time quantitative PCR instrument (Bio-Rad); gel electrophoresis equipment (Beijing Liuyi Instrument Factory); long-wavelength ultraviolet lamp; cell incubator (Thermo), etc.
[0059] 1.2 Materials and reagents: 1kb plus DNA Ladder (invitrogen); Pfu DNA polymerase (Biometics Biotechnology Co., Ltd.); Agarose (BBI); dNTP (Shanghai Sangon Bioengineering Technology Service Co., Ltd.); agarose gel Purification kit (Biometics Biotechnology Co., Ltd.), Lipofectamin TM 2000 (invitrogen), DMEM medium (Gibco), TurboCapture mRNA kit (QIAGEN), SensiMix TM One-Step Kit (Quantace), etc. Other biochemical reagents were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0060] 1.3PCR primers (synthesized by Biomaike Biotechnology Co., Ltd.):
[0061] 5'U6 promoter primer: 5'-AAGGT...
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