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Double-reporter-gene skeleton vector, four-plasmid pseudovirus packaging system and packaged COVID-19 pseudovirus

A dual reporter gene and backbone carrier technology, applied in the fields of genetic engineering and molecular biology, can solve problems such as biological safety hazards and weak basic research

Active Publication Date: 2020-08-28
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the basic research on SARS-CoV-2 is relatively weak, and there are few epidemiological investigations, and these studies generally require the manipulation of live SARS-CoV-2 viruses, and the manipulation of live SARS-CoV-2 viruses has strong biological safety hazards Therefore, it is of great significance to package and prepare pseudoviruses of SARS-CoV-2 with lower biosafety levels

Method used

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  • Double-reporter-gene skeleton vector, four-plasmid pseudovirus packaging system and packaged COVID-19 pseudovirus
  • Double-reporter-gene skeleton vector, four-plasmid pseudovirus packaging system and packaged COVID-19 pseudovirus
  • Double-reporter-gene skeleton vector, four-plasmid pseudovirus packaging system and packaged COVID-19 pseudovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of pCAGGS-SARS-CoV-2-S envelope plasmid

[0035] According to the S gene sequence of SARS-CoV-2 released by NCBI, the codon optimization design facilitates the more efficient expression of the target gene in cells, and the synthesized target gene sequences were respectively synthesized by Nanjing GenScript into the pCDNA3.1 vector After amplifying the target gene by PCR, the target band was recovered and purified by the fragment purification kit, and the fragment and the pVSV-GFP vector were digested with restriction endonucleases (MCS1 (Xhol) and MCS2 (Nhel)) at 37°C After 3h, the carrier and the target fragment were recovered by gel, followed by ligation reaction, and then transferred to competent cells, positive clones were screened by bacterial liquid PCR, enzyme digestion and sequencing were verified to identify the construction of the plasmid (and the constructed plasmid was named pCAGGS-SARS-CoV- 2-S), the specific implementation steps are ...

Embodiment 2

[0062] Example 2 Establishment of pDLV19-mGFP-FLuc pseudovirus packaging system

[0063] In order to avoid the biological safety hazards of traditional live viruses in neutralizing antibody detection experiments, the present invention discloses a pseudovirus packaging system based on four plasmids and double reporter genes. The enhanced green fluorescence of mGFP helps to improve detection sensitivity At the same time, it shortens the cumbersome detection of luc cold fluorescence activity and saves operation time. This technology uses the pDLV19-mGFP-FLuc backbone vector as the core of the dual reporter packaging plasmid expressing FLuc and mGFP, and psl4-gag-pol and psl5-rev As an auxiliary plasmid, use the constructed psl3-S (including its truncated mutant) as the envelope packaging plasmid to perform transient transduction of host cells in vitro at a certain ratio, and collect the supernatant to obtain the new coronavirus pseudovirus. The specific operations are as follows: ...

Embodiment 3

[0073] Example 3 Comparison experiment of pDLV19-mGFP-Fluc pseudovirus packaging system

[0074] By using pDLV19-mGFP-Fluc as the reporter plasmid of the backbone and pDLV19-GFP-luc as the reporter plasmid of the backbone, carry out pseudovirus packaging and infection according to Example 2, observe GFP fluorescence expression and Luc fluorescence intensity, according to image 3 The results showed that the GFP ( image 3 A) and Luc( image 3 B) Compared with pDLV19-GFP-luc (a control pseudovirus packaged in a non-modified vector), the fluorescence expression intensity is stronger after infection, which further improves the detection sensitivity and is more convenient for the naked eye to directly determine the qualitative results of the experiment Analysis, compared with the control, pDLV19-mGFP-Fluc has a higher proportion of positive values ​​for luc, and the difference between groups is more significant in quantitative analysis.

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Abstract

The invention discloses a double-reporter-gene skeleton vector. Double reporter genes of the double-reporter-gene skeleton vector are selected from luciferase and fluorescein, the fluorescein reportergene is preferably an mGFP gene, and the luciferase reporter gene is preferably a Fluc gene. A double-reporter-gene four-plasmid pseudovirus packaging system comprises the double-reporter-gene skeleton vector and three eukaryotic expression packaging plasmids which are respectively integrated with a slow virus gag-pol gene, a slow virus rev gene and an envelope protein gene containing a target virus. A packaged COVID-19 pseudovirus comprises the double-reporter-gene four-plasmid pseudovirus packaging system and a host cell, wherein the host cell is preferably selected from 293T-hACE2 cells. According to the invention, SARS-CoV-2 is specifically implemented, the pseudovirus with single-cycle infection and higher safety can be rapidly packaged, and the vector and the system can be used forresearching coronaviruses such as SARS-Cov2 and the like, provide a powerful screening tool for evaluation of antiviral preparations and vaccines, and have wide application value.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, specifically a double reporter gene backbone carrier, a four-plasmid pseudovirus packaging system, and a packaging pseudovirus for new coronary pneumonia. Background technique [0002] Coronavirus (Coronavirus) belongs to the order Nidovirals (order Nidovirals), Coronaviridae (family Coronavirade), and members of the genus Coronavirus (genus Coronavirus) in virological classification. The genome is single-stranded, positive-strand RNA, and the genome is complete. The length is between 26 and 32 kb, and it is the RNA virus with the largest genome known so far. Coronavirus infection is generally very widespread in nature, and common mammals such as dogs, cats, mice, pigs, cattle and poultry are susceptible. In recent years, various types of coronaviruses have been isolated from beluga whales, camels, and especially bats. There are currently six known human coronaviruses, ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N7/00C12R1/93
CPCC12N15/86C12N15/65C12N7/00C12N2740/15043C12N2800/107C12N2770/20052C12N2770/20021
Inventor 吴飞秦晓峰韦治明杜晓红杨仁祥
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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