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siRNA (Small interfering Ribonucleic Acid) molecule for interfering Survivin expression and application thereof

A molecular and sequence technology, applied in the field of siRNA molecules, can solve problems such as no expression

Active Publication Date: 2011-01-05
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been found that Survivin gene is generally expressed in embryonic tissues and malignant tumors, such as breast cancer, gastric cancer, kidney cancer, melanoma, intestinal cancer, neuroblastoma and ovarian cancer; but in differentiated and mature tissues (except placenta, Proliferative phase endometrium, secretory phase endometrium have a small amount of expression) and no expression in adjacent normal tissues

Method used

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  • siRNA (Small interfering Ribonucleic Acid) molecule for interfering Survivin expression and application thereof
  • siRNA (Small interfering Ribonucleic Acid) molecule for interfering Survivin expression and application thereof
  • siRNA (Small interfering Ribonucleic Acid) molecule for interfering Survivin expression and application thereof

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preparation example Construction

[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 medicament of the present invention on liver cancer is particularly obvious.

[0026] As an 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 p...

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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Abstract

The invention relates to a double-stranded siRNA molecule capable of interfering Survivin gene expression and application thereof to preparing an antitumor medicament. The positive-sense strand of the siRNA molecule is shown as SEQ ID NO:2, and the antisense strand thereof is shown as SEQ ID NO:3, wherein the antisense strand can be specifically combined with the mRNA (Messenger Ribonucleic Acid)of a Survivin gene for degrading the mRNA so as to interfere the translation process after transcription and achieve the purpose of treating tumor.

Description

technical field [0001] The invention relates to a siRNA molecule capable of interfering with the expression of Survivin and its application in the preparation of antitumor drugs. Background technique [0002] Survivin (Survivin) is a new member of the Inhibitorofapoptosis (IAP) family, and is the strongest apoptosis inhibitor found so far. Survivin has complex functions, inhibits apoptosis, promotes cell transformation and participates in cell mitosis, angiogenesis and tumor cell drug resistance. The Survivin gene was screened and cloned in the human genome library by Ambrosini et al. (Nature Med, 1997, 3(8): 917-921). The gene is 15KB in length, located at 17q25, and contains 4 exons and 3 introns. Contains, Survivin gene encoding product is a protein composed of 142 amino acids, molecular weight 16.2KD. Studies have shown that the high expression of Survivin can inhibit the apoptosis induced by various factors such as p53 and caspases. It has been confirmed by transgeni...

Claims

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Application Information

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IPC IPC(8): C12N15/11A61K48/00A61P35/00A61P35/02
Inventor 孙云成陈莉李铁军陆毅祥朱远源
Owner BIOMICS BIOTECH
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