Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF4 Cites 4 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com