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Double-strand siRNA inhibiting survivin gene expression, application thereof and expression plasmid and transfersome containing same

A technology for gene expression and expression plasmid, which is applied in the field of biomedicine to achieve the effect of improving the hit rate

Inactive Publication Date: 2015-12-23
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double-stranded siRNA molecules have technical advantages such as high specificity and high efficiency, but there are still some technical problems to be solved in the clinical application of double-stranded siRNA molecular drugs

Method used

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  • Double-strand siRNA inhibiting survivin gene expression, application thereof and expression plasmid and transfersome containing same
  • Double-strand siRNA inhibiting survivin gene expression, application thereof and expression plasmid and transfersome containing same
  • Double-strand siRNA inhibiting survivin gene expression, application thereof and expression plasmid and transfersome containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Detection experiment of chemically synthesized double-stranded siRNA molecules on the silencing effect of survivin gene

[0043] 1. Main instruments, reagents and materials.

[0044] 1.1 Instruments: nucleic acid synthesizer (GE Company), PCR instrument (ABI Company); real-time quantitative PCR instrument (Bio-Rad); cell incubator (Thermo), etc.

[0045] 1.2 Materials and reagents: RNAiMAXTM (invitrogen), 1640 medium (Gibco),

[0046] TurboCapture mRNAkit (QIAGEN), SensiMix TM one-StepKit (Quantace), NT-siRNA sense strand: (5'-CCACCGUGUCUCAGUCCUAdTdT-3'; antisense strand: 5'-UAGGACUGAGACACGGUGGUA-3') and the like.

[0047] 1.3 PCR primers (synthesized by Biomaike Biotechnology Co., Ltd.):

[0048] Survivin forward primer: 5'-GGGCAACGAGGGGCACCATC-3'

[0049] Survivin reverse primer: 5'-AACACGCGGCACGGTGCGTGA-3'

[0050] GAPDH forward primer; 5'-GCTCTCTCCTGCTCCTGTTC-3'

[0051] GAPDH reverse primer: 5'-ACGTCGAAATCCGTACACGC-3'

[0052] 2. Chemical synthe...

Embodiment 2

[0068] Example 2: Antitumor activity experiments of cationic liposomes encapsulating double-stranded siRNA molecules on A549 and PC3 cells

[0069] 1 reagent

[0070] Dioleoyl phosphatidyl ethanol (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine, DOPE) (German lipoid company), cholesterol, methoxylated polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine (DSPE- mPEG2000) (lipoid company in Germany), chloroform, sucrose (China Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd.), DOTAP (Biontex, Germany)

[0071] 2 instruments

[0072] LF-1 film extruder: Canadian AVESTIN company

[0073] N-1001D-WA Rotary Evaporator: Tokyo Rika, Japan

[0074] Y92-ⅡN Ultrasonic Cell Pulverizer: Ningbo Xinzhi Biotechnology Co., Ltd.

[0075] -80°C low temperature refrigerator: REVCO, USA

[0076] Milli-QUFPLUS water purifier: Millipore, USA

[0077] Autoclave TH-3560: Zaoxin (Xinke) Enterprise Co., Ltd.

[0078] 3 Preparation of liposomes encapsulating double-stranded siRNA m...

Embodiment 3

[0087] Example 3: Antitumor activity of 2'-methoxy-modified double-stranded siRNA molecules on A549 and PC3 cells

[0088] 1 The instruments and reagents used are the same as those in Example 1

[0089] Preparation of 22'-methoxy-modified double-stranded siRNA molecules

[0090] The nucleotides of the double-stranded siRNA molecules obtained in step 2 of Example 1 were modified with a methoxyl group of the 2'-sugar ring. According to the design idea, 1-10 deoxyribose sugars at the 3' end of the sense strand of the above-mentioned double-stranded siRNA molecules were modified. 2'-methoxy group was modified at different sites, and Guangzhou Ribo Biotechnology Co., Ltd. was entrusted to modify the above siRNA to obtain a series of double-stranded siRNA molecular sequences, from which the 16th nucleotide with the strongest activity was screened. Sequence 2'-methoxysugar ring chemically modifies double-stranded siRNA. Tumor cell proliferation and protein expression inhibition exp...

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Abstract

The invention discloses a siRNA sequence relating to inhibition of survivin gene expression. The siRNA sequence is composed of a positive-sense strand and an anti-sense strand which have the following nucleotide sequences, the positive-sense strand is 5'-GGCCCUUGUCUAAGUGCAA-3', and the anti-sense strand is 5'-UUGCACUUAGACAAGGGCC-3'. The siRNA sequence has broad-spectrum siRNA antitumor effect, and has good inhibiting effect on breast cancer, lung cancer, prostate cancer, intestinal cancer, liver cancer, leukemia, stomach cancer, cervical cancer and oral epithelial cancer in vitro, especially for lung cancer and prostate cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a double-stranded siRNA molecule for inhibiting the expression of survivin gene and its application in the preparation of antitumor drugs, including an expression plasmid and a transfer body thereof. Background technique [0002] Malignant tumors are one of the main killers that threaten human life and health. Although various treatment methods are constantly emerging, there is no truly effective cure method yet. The treatment of tumors has always been a worldwide problem that has long plagued the medical community. Evasion of apoptosis is a hallmark of all tumors, allowing tumor cells to survive abnormal growth stimuli and resist chemotherapy and radiotherapy. The anti-apoptotic protein survivin inhibits the growth of tumor cells by interacting with apoptotic signaling and becoming the new most potential therapeutic target. [0003] Apoptosis suppressor gene su...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85A61K48/00A61K31/713A61P35/00A61P35/02
Inventor 滕乐生郝斐李剑光谢晶孟庆繁逯家辉刘艳刘洋权宇彤
Owner JILIN UNIV
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