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Pseudo-virus packaging system of coronavirus and one-step packaging method

A coronavirus, packaging system technology, applied in the fields of genetic engineering and virology, can solve problems such as weakness and hidden dangers of biological safety, and achieve the effect of high titer, easy operation and low background value

Inactive Publication Date: 2020-11-06
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the basic research on COVID-19 is relatively weak, and there are few epidemiological investigations. Existing studies have shown that the transmission capacity of COVID-19 transmitted by aerosol is more than ten times that of SARS. Therefore, it is necessary to operate COVID-19 in non-P3 laboratories. Live viruses pose a great biological safety hazard

Method used

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  • Pseudo-virus packaging system of coronavirus and one-step packaging method
  • Pseudo-virus packaging system of coronavirus and one-step packaging method
  • Pseudo-virus packaging system of coronavirus and one-step packaging method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of different types of envelope plasmids:

[0047] 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 vector 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 COVI...

Embodiment 2

[0080] Example 2 The infection of VSV-COVID-19-S-C19-HA on 293T-hACE2 cells showed higher efficiency:

[0081] In order to obtain VSV pseudoviruses of different truncated spike proteins (S) 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), which is a VSV replication-deficient virus strain, into 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 plasmid was transiently transfected 12 hours in advance), the supernatant was collected, and anti-VSV-G neutralizing serum was added to block the residual dVSVΔG-Fluc-EGFP in the cell supernatant The infectivity of the progeny virus was obtained by harvesting, and t...

Embodiment 3

[0084] Example 3 The titer of dVSVΔG-COVID-19-S-C19-HA pseudovirus packaged in 293T cells is the highest

[0085] 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:

[0086] 1) Vero / BHK21 / 293T / 293T-hACE2 cells are plated in 6-well plates, and the cell density should be around 70% after 24 hours;

[0087] 2) pCAGGS-S / pCAGG...

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Abstract

A pseudo-virus packaging system of coronaviruses comprises a vesicular stomatitis virus (VSV) vector with a Fluc and EGFP double reporter gene replacing a GP gene and an assembly cell expressing coronavirus spike protein S. The double reporter gene is selected from luciferase and fluorescent protein, and the luciferase reporter gene is preferably a Fluc gene. The fluorescent protein reporter geneis preferably an EGFP gene, and the assembly cell is preferably 293T. The invention further relates to a one-step packaging method of the packaging system; a pseudo-virus with single-cycle infection,low background value and high titer can be quickly packaged through simple, convenient and rapid operation, and the packaging system has the characteristic of being rapid in detection compared with aslow virus mediated pseudo-virus system; the one-step packaging method can be used for researching COVID-19, SARS, MERS and other coronaviruses and other viruses, provides a powerful screening tool for evaluation of antiviral preparations and vaccine, and has wide application value.

Description

technical field [0001] The invention relates to the fields of genetic engineering and virology, in particular to a pseudovirus packaging carrier and a packaging system, which are used for a one-step packaging method for preparing a coronavirus pseudovirus. 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-NL63, and HCoV-229E. To date, there are no approved treatments or vaccines for human infection with the coronavirus. [0003] Cell assembly during coronavirus infection is mediated by the transmembrane spike (S) glycoprotein, which forms homotrimers protruding from the surface of the virus wit...

Claims

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Application Information

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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 秦晓峰吴飞韦治明龙丽梅杨仁祥
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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