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siRNA for inhibiting expression of K-RAS genes as well as precursor and application thereof

一种表达载体、前体的技术，应用在DNA / RNA片段、基因治疗、基因工程等方向，能够解决EGFR靶向药物无效等问题

Inactive Publication Date: 2017-11-14
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, considering that if the EGFR and K-RAS pathways can be targeted at the same time, the upstream and downstream of the pathways can be inhibited at the same time, so that EGFR-targeted drugs can have a better therapeutic effect on patients with K-RAS mutations. Therefore, there is an urgent need for A treatment method that can target and inhibit the K-RAS gene and corresponding drugs to solve the problems that there is no drug specific for K-RAS mutations, and K-RAS mutations cause EGFR-targeted drugs to be ineffective

Method used

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  • siRNA for inhibiting expression of K-RAS genes as well as precursor and application thereof
  • siRNA for inhibiting expression of K-RAS genes as well as precursor and application thereof
  • siRNA for inhibiting expression of K-RAS genes as well as precursor and application thereof

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Experimental program
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Effect test

Embodiment 1

[0131] Embodiment 1 expression vector construction

[0132] For biosafety reasons, the plasmid is first modified. Biotoxic elements such as EmGFP and Blasticidin are excised by DNA restriction enzymes. figure 1 Shown is the plasmid before transformation, figure 2 Shown is the plasmid after excision of EmGFP and Blasticidin.

[0133] Among them, pCMV represents the eukaryotic promoter, pUC ori represents the replication origin site of the plasmid in prokaryotic cells, and the initiation site does not express the inserted sequence, and Spectinomycin represents the spectinomycin resistance gene, which is used for the screening of the plasmid.

[0134] After the plasmid transformation was completed, complementary oligo DNA was designed and synthesized according to the K-RAS gene sequence, and the K-RAS siRNA sequence: 5'-GGUGACUUAGGUUCUAGAU-3' (SEQ ID NO: 263), the sequence is shown in Table 1.

[0135] Table 1. oligo DNA sequences and their corresponding precursor siRNA element...

Embodiment 2

[0147] Example 2 The therapeutic effect of K-RAS siRNA on Lewis lung cancer in mice

[0148] LLC (Lewis lung cancer) cell line was provided by the School of Life Sciences, Nanjing University. DMEM is a product of Hyclone Company. Fetal bovine serum was a product of Gibco. During the experiment, the LLC cell line was completely cultured with DMEM containing 10% FBS, 100ug / ml penicillin and 100ug / ml streptomycin at 37°C, 5% CO 2 cultured in an incubator.

[0149] The experimental animals were 15 C57BL / 6 mice, half male and half male, 6 weeks old, provided by Model Animal Institute of Nanjing University.

[0150] First culture LCC cells, digest the LCC cells cultured to the logarithmic growth phase with trypsin, centrifuge at 1000rpm, discard the supernatant, wash twice with sterile saline, suspend the cells in saline, and observe with trypan blue staining Cell viability, and cell counting, adjust the cell density to 5 × 10 6 pieces / ml. During the test, healthy C57BL / 6 mice...

Embodiment 3

[0162] Example 3 The therapeutic effect of K-RAS siRNA plasmamid on colon cancer in mice

[0163] Colon cancer cell line: mouse colon cancer cell CT-26 (derived from BALB / c, H-2Kd): provided by the School of Life Sciences, Nanjing University.

[0164] Modeling experimental animals: BALB / c mice, female, 6-7 weeks old, provided by Model Animal Institute of Nanjing University.

[0165] Establishment of animal model: BALB / c mice are animals of the same strain as the tumor CT-26 cell line. The revived CT-26 was subcultured. When the cells grow to a certain amount, take the cells in the logarithmic growth phase and add 0.9% saline to adjust the cell concentration to 5×10 6 / ml, according to the dose of 0.2ml per mouse (about 1×10 6 cells / only), the tumor cells were inoculated subcutaneously in the right armpit of the mice, and fed with normal diet after inoculation.

[0166] One week later, all 15 BALB / c tumor-bearing mice had tumors in their axillae, indicating that the model w...

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Abstract

The invention discloses siRNA for inhibiting expression of K-RAS genes as well as a precursor sequence and application thereof. The K-RAS siRNA provided by the invention and the precursor sequence thereof can effectively inhibit expression of the K-RAS genes, and an in vitro experiment proves that the siRNA has a certain inhibition effect on K-RAS high expression tumors. The precursor and a carrier of the siRNA disclosed by the invention can form stable siRNA in a host of the siRNA, and take an effect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an siRNA for inhibiting K-RAS gene expression, its precursor and application. Background technique [0002] RNA interference (RNAi) is a powerful experimental tool in the laboratory, using homologous double-stranded RNA (dsRNA) to induce sequence-specific silencing of target genes and rapidly block gene activity. siRNAs play a central role in the RNA silencing pathway, directing the degradation of specific messenger RNAs (mRNAs). siRNA is an intermediate product in the RNAi pathway and an essential factor for RNAi to exert its effect. The formation of siRNA is mainly regulated by Dicer and Rde-1. Due to RNA virus invasion, transcription of transposons, transcription of inverted repeat sequences in the genome, etc., dsRNA appears in cells, and the protein encoded by Rde-1 (RNAi-deficient gene-1) recognizes exogenous dsRNA. When dsRNA reaches a certain amount At this time,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61P35/00
CPCC12N15/1137C12N2320/30C12N2310/141A61K31/7088C12N2310/14A61P35/00C12N2310/531
Inventor 张辰宇陈熹梁宏伟U·乌尔-拉赫曼曾科
Owner JIANGSU MICROMEDMARK BIOTECH
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