ShRNA for inhibiting replication of SARS-COV-2 virus and application of shRNA

A virus replication, sars-cov-2 technology, applied in DNA/RNA fragments, antiviral agents, recombinant DNA technology, etc., can solve problems such as economic losses

Inactive Publication Date: 2020-12-22
SHENZHEN INT INST FOR BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific treatment or drug for COVID-19, and disease control mainly relies on strict physical isolation to cut off the transmission route, which directly causes serious economic losses

Method used

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  • ShRNA for inhibiting replication of SARS-COV-2 virus and application of shRNA
  • ShRNA for inhibiting replication of SARS-COV-2 virus and application of shRNA
  • ShRNA for inhibiting replication of SARS-COV-2 virus and application of shRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1 Interference effect of shRNA carried by adeno-associated virus

[0147] virus strain

[0148] The novel coronavirus (SARS-CoV-2) strain (GenBank: MT123290) was isolated from a throat swab of a patient and kept in the P3 laboratory of Guangzhou Customs Technical Center.

[0149] Reagent material

[0150] Vero E6 cells, 96-well cell culture plate, DMEM medium, 2% bovine serum DMEM medium, primary antibody, secondary antibody, etc. Freshly prepared 10% hypochlorous acid solution, 4% paraformaldehyde, 1.6% CMC.

[0151] experimental method

[0152] Take Vero E6 cells susceptible to SARS-COV-2, according to 1*10 4 per well into a 96-well plate. After 24 hours of wall attachment, each well was transfected with lipofectamine3000 reagent according to the amount of 0.25 μg shRNA contained in adeno-associated virus. After 24 h, the transfection efficiency was observed.

[0153] Bring the 96-well plate into P3, and add SARS-COV-2 at a dose (non-lethal dose) with a...

Embodiment 2

[0170] Example 2 The interference effect of lentivirus-encapsulated shRNA

[0171] experimental method

[0172] Infect the Vero E6 cells with the packaged lentivirus respectively, and select the stably transfected cells by puromycin resistance, according to 1.5*10 4 One per well was placed in a 96-well plate, and after 24 hours of attachment, SARS-COV-2 was added at a dose (non-lethal dose) of 0.05 at the multiplicity of infection (MOI). The infection efficiency was determined by immunofluorescence (as described in Example 1).

[0173] Experimental results

[0174] Such as image 3 and 4 As shown, compared with the control, the interference effects of shRNA5, shRNA6, shRNA7, shRNA9, and shRNA10 carried by the lentivirus are extremely significant.

Embodiment 3

[0175] Example 3 Effect of shRNA on viral replication

[0176] experimental method

[0177] Vero E6 cell lines stably transfected with different shRNAs were used at 1×10 per well 5 Cells were plated in 24-well plates, with 4 replicates of each shRNA, and GFP was used as a control. Inoculate SARS-CoV-2 according to MOI=0.05, 24 hours after infection, scrape off the cell culture supernatant and cells together, freeze and thaw once, and use Focus-forming Assay (FFA) to measure the virus in the cell culture Titer. Specific steps are as follows:

[0178] 1. Seed VeroE6 cells in 96-well flat-bottom plate, 2×104 cells / well.

[0179] 2. Wait until the cells reach 100% confluence

[0180] (1) Take 20 μl of the virus solution to be tested, serially dilute it 10 times with DMEM containing 2% FBS, and mix well.

[0181](2) Discard the cell culture medium, add virus supernatants of different dilutions to the cell wells, 50 µL / well, shake slightly to make the liquid evenly cover the b...

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Abstract

The invention discloses shRNA for inhibiting replication of an SARS-COV-2 virus and an application of the shRNA. The shRNA targets one of E, M and N genes of the SARS-COV-2 virus and comprises any onepair of sequences selected from the following combinations: (a) a reverse repetitive sequence pair consisting of complementary SEQ ID NO: 1 and SEQ ID NO: 2; (b) a reverse repetitive sequence pair consisting of complementary SEQ ID NO: 3 and SEQ ID NO: 4; (c) a reverse repetitive sequence pair consisting of complementary SEQ ID NO: 5 and SEQ ID NO: 6; (d) a reverse repetitive sequence pair consisting of complementary SEQ ID NO: 7 and SEQ ID NO: 8; and (e) a reverse repetitive sequence pair consisting of complementary SEQ ID NO: 9 and SEQ ID NO: 10. The invention also discloses a drug for inhibiting the replication of the SARS-CoV-2 virus in a subject. The drug comprises a vector and a nucleic acid sequence encoding one or more of the shRNAs.

Description

technical field [0001] The present invention relates to a kind of shRNA (short hairpin RNA), in particular to a kind of shRNA for inhibiting the replication of SARS-COV-2 virus and its application. Background technique [0002] Corona Virus Disease 2019 (COVID-19), referred to as "New Coronary Pneumonia", is the most extensive global pandemic that humans have encountered in the past century, posing a huge threat to global public health security. At present, there is no specific treatment or drug for COVID-19, and disease control mainly relies on strict physical isolation to cut off the transmission route, which directly causes serious economic losses. [0003] For severe and critical cases, the following methods can be used for treatment: (1) Respiratory support, including oxygen therapy, high-flow nasal cannula oxygen therapy or non-invasive mechanical ventilation, invasive mechanical ventilation and rescue therapy; (2) Circulatory support, adequate On the basis of fluid r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P31/14
CPCC12N15/1131A61K31/713A61P31/14C12N2310/141C12N2310/531
Inventor 苏金赵金存杨鹏辉孙静苗凯
Owner SHENZHEN INT INST FOR BIOMEDICAL RES
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