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Coronavirus pseudovirus packaging system and packaging method, and application of coronavirus pseudovirus to evaluating disinfection efficacy

A coronavirus and packaging system technology, applied in the direction of applications, viruses, and viral peptides, can solve the problems of lack of evaluation methods for the ability of disinfectants to inactivate viruses, uneven virus inactivation capabilities, and lack of data support in the scope of application. Fast, easy to operate, high biosafety results

Active Publication Date: 2021-05-07
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common virus killers include ozone, ultraviolet light, chlorine dioxide, peracetic acid, hydrogen peroxide, sodium dichloroisocyanurate, irradiation, and negative ions. , the main reason is the lack of a unified and effective method for evaluating the ability of disinfectants to inactivate viruses
Especially for highly transmissible and harmful viruses such as the new crown (SARS-CoV-2), acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), live Virus evaluation can only choose P3 laboratory operation
The lack of test and evaluation methods with biological safety directly leads to the lack of evaluation support for applicable new technologies or new applications such as ozone and irradiation, which affects the determination of its parameters, applicable rules, and popularization and application.

Method used

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  • Coronavirus pseudovirus packaging system and packaging method, and application of coronavirus pseudovirus to evaluating disinfection efficacy
  • Coronavirus pseudovirus packaging system and packaging method, and application of coronavirus pseudovirus to evaluating disinfection efficacy
  • Coronavirus pseudovirus packaging system and packaging method, and application of coronavirus pseudovirus to evaluating disinfection efficacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Construction of different types of envelope plasmids:

[0066] According to the codon-optimized sequence of the S gene released by NCBI to facilitate its expression in cells, the sequences were synthesized by Nanjing GenScript Biotechnology into pCDNA3.1 vectors, and the target genes were amplified by PCR and recovered by fragment purification kits Purify the target band, digest the fragment and the pCAGGS vector with restriction endonucleases MCS1 (Xhol) and MCS2 (Nhel) at 37°C for 3 hours, recover the carrier and the target fragment from the gel, perform a ligation reaction, and then transfer to a competent state Cells and bacterial liquid PCR screened positive clones and enzyme digestion and sequencing to verify and identify the construction of the plasmid. The specific implementation steps are as follows: 1. Primer synthesis and primer information: The primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., which was constructed to amplify COV...

Embodiment 2

[0099] Example 2 The infection of VSV-COVID-19-S-C19-HA on 293T-hACE2 cells showed higher efficiency:

[0100] In order to obtain the VSV pseudovirus of the spike protein (S) of different truncated forms of COVID-19, plasmids pCAGGS-COVID-19-S, pCAGGS-COVID-19-S-C19, pCAGGS-COVID-19-S-C19- HA, pCAGGS-COVID-19-S-C27, and pCAGGS-COVID-19-S-C53 were transfected into 293T cells by liposomes (lipo2000). 12 hours after transfection, inoculate dVSVΔG-Fluc-EGFP (preserved in the laboratory), i.e., VSV replication-defective virus strains, into the cells expressing the complete spike protein of COVID-19 or COVID-19-S-C19 / C27 / C53 / C19 respectively. -In the medium corresponding to the cells of the HA truncated protein (eukaryotic expression plasmids were transiently transformed 12h in advance), the supernatant was collected, and anti-VSV-G neutralizing serum was added to block the residual dVSVΔG-Fluc- in the cell supernatant. The infectivity of EGFP, the progeny virus was harvested, and ...

Embodiment 3

[0103] Example 3 The titer of dVSVΔG-COVID-19-S-C19-HA pseudovirus packaged in 293T cells is the highest

[0104]The packaging efficiency of the new coronavirus pseudovirus is one of the main limiting factors for high-throughput detection of neutralizing antibody tests in vitro. In order to select the most suitable cell line for the production of 2019-nCoV pseudotyped virus, conventional cells such as Vero-E6, BHK21, 293T-hACE2 and 293 were compared in this technique by pre-plating different cell lines in 6-well cell culture plates, The first choice is to transfect plasmids of different concentrations in the above-mentioned different cell lines, and then refer to the following one-step packaging method to package the dVSVΔG-COVID-19-S-C19-HA new coronavirus pseudovirus. The specific implementation steps are as follows:

[0105] 1) Vero / BHK21 / 293T / 293T-hACE2 cells were plated in a 6-well plate, and the cell density should be around 70% after 24 hours;

[0106] 2) pCAGGS-S / pCAG...

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Abstract

The invention relates to a coronavirus pseudovirus packaging system. The coronavirus pseudovirus packaging system comprises a vesicular stomatitis virus VSV vector and an assembly cell, wherein the vesicular stomatitis virus VSV vector is formed by replacing GP genes with Fluc and EGFP double reporter genes, and the assembly cell is used for expressing coronavirus spike protein S. The double reporter genes are selected from luciferase and fluorescent protein, and the luciferase reporter gene is preferably the Fluc gene. According to the packaging system, a one-step packaging method is adopted, so that pseudoviruses which are infected in a single cycle, low in background value and high in titer and have the characteristic of rapid detection compared with a lentivirus-mediated pseudovirus system can be rapidly packaged, and the packaging system can be used for researching coronaviruses such as COVID-19 (SARS-CoV-2), SARS (SARS-CoV) and MERS; and the pseudoviruses can be used for evaluating the efficacy of a disinfectant through the steps of a virus pollution distribution model, scene building and sampling detection, a safe, convenient and effective tool method is provided for evaluating the disinfectant, and the pseudoviruses have wide application value.

Description

technical field [0001] The present invention relates to the fields of gene and cell engineering and virology, in particular to a pseudovirus packaging carrier and a packaging cell system. The pseudovirus packaging carrier and the packaging system prepare a coronavirus pseudovirus through a one-step packaging method, and the prepared coronavirus pseudovirus It can be used as a biological indicator to detect and evaluate the effectiveness of biological, chemical substances and physical treatment methods for inhibiting and disinfecting coronaviruses. Background technique [0002] COVID-19 is a virus that can infect humans with deadly pneumonia. In addition to 2019-nCoV, two other coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), have also caused fatal disease in humans since the early 2000s. pneumonia. In addition, four low-pathogenic coronaviruses circulate in humans: HCoV-OC43, HCoV-HKU1, HCoV...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/40C12N5/10C12N15/867C12N15/65G01N33/569
CPCC12N7/00C12N15/86C12N15/65C07K14/005G01N33/56983C12N2760/20221C12N2770/20022C12N2770/20021C12N2740/15043Y02A50/30A01N63/40A01P1/00C12N2760/20242C12N2770/20041
Inventor 秦晓峰韦治明龙丽梅杨仁祥李帆
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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