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Novel coronavirus SARS-CoV-2 safe replication subsystem and application thereof

A coronavirus, sars-cov-2 technology, applied in the direction of viruses, applications, viral peptides, etc., can solve problems such as time-consuming, labor-intensive, large molecular weight, and lack of expression level

Active Publication Date: 2020-12-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the system is based on the BAC plasmid
The BAC plasmid has a large molecular weight and is unstable. It cannot reach the ideal expression level after transducing the cells. At the same time, it is time-consuming and laborious to operate.

Method used

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  • Novel coronavirus SARS-CoV-2 safe replication subsystem and application thereof
  • Novel coronavirus SARS-CoV-2 safe replication subsystem and application thereof
  • Novel coronavirus SARS-CoV-2 safe replication subsystem and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Construction of Example 1 Replicon

[0099] Based on the genome composition of the new coronavirus and the principle process of viral RNA synthesis (replication and transcription process), the inventor's team creatively constructed a safe replicon of the new coronavirus SARS-CoV-2, including the following two expression structures :

[0100] (I) encoding the non-structural protein of the novel coronavirus SARS-CoV-2;

[0101] (II) The 5'UTR and 3'UTR of the new coronavirus SARS-CoV-2, and the transcriptional regulatory regions and reporter genes that the non-structural proteins of the new coronavirus SARS-CoV-2 can act on.

[0102] (I) The non-structural protein encoding the novel coronavirus SARS-CoV-2 is an expression vector encoding the nsp1-nsp16 protein sequence.

[0103] The sequences of rep1a and rep1b in the genome of the new coronavirus total about 20,000 bp, accounting for about 2 / 3 of the virus genome. Considering the efficiency of transfection and express...

Embodiment 2

[0174] The establishment of embodiment 2 novel coronavirus SARS-CoV-2 replicon system

[0175] The purpose of constructing the replicon system in Example 1 is to screen anti-new coronavirus SARS-CoV-2 drugs, especially human drugs, so the HEK 293T cell line was selected as packaging cells for verification. The schematic diagram of the working principle of ps2V, ps2AN, ps2AC, ps2B4 expression vectors in human body or human cells is attached Image 6 shown.

[0176] HEK293T cells in good growth state were evenly spread in 12-well culture plates treated with poly-lysine (cell density was about 6.5×10 4 / cm 2 ), the cells are required to be single and evenly distributed. After about 24 hours of culture, the cell confluency should be close to 80%. At this point, the Opti-Lipo2000-DNA mixture was prepared as shown in Table 1 for transfection.

[0177] The concentration of the four carriers can be in the range of 0.01-1 μg / μL, and the ratio of the four carriers can be adjusted w...

Embodiment 3

[0182] Embodiment 3 detects the performance of the new coronavirus SARS-CoV-2 replicon system

[0183] The ps2V, ps2AN, ps2AC, and ps2B plasmids were transfected according to the steps in Example 2. 6h after transfection, Remdesivir (Remdesivir), lopinavir ( Lopinavir), Ritonavir (Ritonavir). After 24 hours of drug treatment, the cell luciferase activity was detected, and the inhibition rate was calculated based on the DMSO control, and the half-inhibitory concentration of the drug (hereinafter referred to as IC50) was calculated using GraphpadPrism 7.0 software. For specific results, see Figures 10 to 12 .

[0184] in Figure 10 The results showed that the IC50 of Remdesivir was 12.4±1.08μM; Figure 11 The results showed that the IC50 of Lopinavir was 6.785±1.09μM; Figure 12 The results showed that the IC50 of Ritonavir was 14.77±1.05 μM.

[0185] The above data results show that the replicon system constructed in Example 1 can reproduce the response of wild-type SAR...

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Abstract

The invention discloses a novel coronavirus SARS-CoV-2 safe replication subsystem and application thereof in screening anti-SARS-CoV-2 drug virus drugs. The novel coronavirus SARS-CoV-2 safe replication subsystem specifically comprises a non-structural protein for encoding novel coronavirus SARS-CoV-2, 5' UTR and 3' UTR of the novel coronavirus SARS-CoV-2, a transcriptional regulation area and a reporter gene, wherein the transcriptional regulation area and the reporter gene can be acted by the non-structural protein of the novel coronavirus SARS-CoV-2. By means of the SARS-CoV-2 safe replication subsystem, high-throughput screening of the anti-SARS-CoV-2 drugs and drug efficacy verification of the drugs can be conducted under the condition that the SARS-CoV-2 safe replication subsystem does not depend on a biosafety three-level laboratory, and operation is easy and convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a safe replicator system of a novel coronavirus SARS-CoV-2 and an application thereof. Background technique [0002] As of July 23, 2020, the new coronavirus SARS-CoV-2 has infected more than 15 million people worldwide and killed more than 140,000 people. However, the current clinical therapeutic drugs applied to SARS-CoV-2 infection are very limited. For reasons of biosafety, the development and screening of drugs against wild-type SARS-CoV-2 can only be carried out in biosafety level 3 laboratories (P3 laboratories), which greatly limits the antiviral agents against SARS-CoV-2 drug development. [0003] Previous studies have also shown that by inserting the E protein-deleted coronavirus genome into the artificial chromosome (Bacterial Artificial Chromosome, BAC), the constructed safe replicator system can simulate the replication of coronavirus. This system has ...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/63C12N15/65C12N5/10C12Q1/02C12Q1/66
CPCC07K14/005C12N15/63C12N15/65C12Q1/66G01N33/5008C12N2770/20022C12N2770/20052G01N2500/10
Inventor 张辉罗越文
Owner SUN YAT SEN UNIV
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