Small interfering RNA targeting human ran and its use in the preparation of anti-hepatitis C medicaments

A small interference, drug technology, applied in the field of medicine, can solve the problem of not being able to block the transmission of HCV

Inactive Publication Date: 2019-02-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no HCV preventive vaccine in the world, which cannot block the transmission of HCV

Method used

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  • Small interfering RNA targeting human ran and its use in the preparation of anti-hepatitis C medicaments
  • Small interfering RNA targeting human ran and its use in the preparation of anti-hepatitis C medicaments
  • Small interfering RNA targeting human ran and its use in the preparation of anti-hepatitis C medicaments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Analysis of RAN expression level after JC1 infection

[0027] Establishment of HCV Cell Infection Model

[0028] 1 day before transfection, with 1×10 7 Inoculate a 10 cm culture dish at a density of cells / well, and the degree of cell confluence is 70% to 80%. During transfection, 10 μg of HCV RNA transcripts were introduced into the liver cancer cell line Huh7.5.1 supporting HCV replication by electrotransfection with a single square wave at 260V and a pulse length of 25 milliseconds. The cell supernatant was collected, centrifuged at 1500 g for 5 min to remove cell debris, and stored at -70°C. Replant Huh7.5.1 cells in a six-well cell culture plate. When the cells reach 50% to 60% confluence, add 2ml of the collected cell supernatant to each well, incubate for 24 hours, wash with PBS three times, and add complete medium to continue culturing . Cells were harvested 48 hours later for subsequent experiments.

[0029] Fluorescent quantitative PCR

[0030] ...

Embodiment 2

[0034] Example 2 Screening of RAN interference sequences

[0035] RNA interference

[0036] 1 day before transfection, with 2×10 5 6-well cell culture plates were inoculated at a density of cells / well. For transfection, take 10 μl siRNA (commissioned by Ribo Biosynthesis) and dilute it with 100 μl Opti-MEM I serum-free medium, mix gently; take 2 μl Lipofectamine TM For RNAiMAX, dilute in 100μl Opti-MEM I serum-free medium and mix gently. Diluted HCV RNA and Lipofectamine TM RNAiMAX was mixed gently and allowed to stand at room temperature for 20 minutes. Add 200 μl of the complex to the cells in the 6-well plate and mix gently. After 24 hours, add 2ml of JC1 virus supernatant to each well to infect the cells, and after incubation for 24 hours, add complete medium to continue culturing. Cells were harvested 48 hours later for subsequent experiments.

[0037] Fluorescent quantitative PCR

[0038] Fluorescent quantitative PCR was used to detect the relative amount of HCV ...

Embodiment 3

[0042] Embodiment 3 003 anti-HCV effect analysis

[0043] Western Blot detection

[0044] The expression level of HCV core protein was detected by Western Blot. After RNA interference, the total cellular protein was extracted and quantitatively analyzed according to the operating instructions of the BCA protein quantification kit. 40 μg of the above-mentioned total cellular protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk powder, the membrane was incubated with antibodies HCV Core (Abcam) and β-actin (Cellsignaling Technology) at 4°C overnight, and the secondary antibody was incubated for 1 hour and detected with chemiluminescence reagent (Pierce).

[0045] Such as image 3 As shown, 003 can significantly inhibit the expression of HCV core protein.

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Abstract

The invention relates to the technical field of medicine, particularly relates to small interfering RNA (ribose nucleic acid), in particular to the small interfering RNA aiming at an RAN (ras-related nuclear protein) gene target and an application of the small interfering RNA in preparation of medicine for treating genotype 2 HCV. The sequence of a positive-sense strand of small interfering RNA of targeting human RAN is ACAGGAAAGUGAAGGCGAA dTdT, and the sequence of an antisense strand is dTdT UGUCCUUUCACUUCCGCUU. The experiment proves that the si RNA interference sequence 003 of targeting human RAN is transfected to Huh7.5.1 cells, expression of RAN genes in the cells is silenced, nucleus-cytoplasm translocation of PTB (polypyrimidine tract binding) can be inhibited, and accordingly, the anti-HCV effect can be developed. Therefore, RAN can be taken as the molecular target, the si RNA interference sequence 003 of RAN is taken as an active ingredient for preparation of the medicine, and the medicine is used for treatment of HCV.

Description

technical field [0001] The present invention relates to the field of medical technology, in particular to small interfering RNAs, in particular to small interfering RNAs targeting RAN (Ras-related nuclear protein) genes and their use in the preparation of drugs for treating genotype 2 hepatitis C virus . Background technique [0002] Hepatitis C virus (HCV) infection can cause acute / chronic hepatitis, liver cirrhosis and liver cancer. At present, there are about 185 million HCV-infected people in the world, and the infection rate is about 1-2%, and the infection rate in East Asia is 3.7% (World Health Organization 2014 Hepatitis C Diagnosis and Treatment Guidelines). There are more than 3 million newly infected people every year, especially in developing countries. About 500,000 HCV-infected people die each year. Existing treatments can only achieve sustained virological response (SVR) in about 50% of patients, and there are problems such as contraindications, individual ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P31/14
Inventor 薛继华陈智吴珊珊朱海红刘艳宁
Owner ZHEJIANG UNIV
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