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Viral structural protein genetic complement based coronavirus cell model

A coronavirus and structural protein technology, applied in the field of biomedicine, can solve problems such as large genome fragments, inability to accommodate plasmids, and unstable clones

Pending Publication Date: 2021-05-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty in realizing the rescue of coronavirus is that its genome fragment is too large to be accommodated by conventional genetic engineering plasmids, and an alternative vector needs to be found; in addition, there is also the problem of "unstable cloning"

Method used

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  • Viral structural protein genetic complement based coronavirus cell model
  • Viral structural protein genetic complement based coronavirus cell model
  • Viral structural protein genetic complement based coronavirus cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The following describes exemplary embodiments of the present invention by taking the Wuhan-Hu-1 strain (MN908947) of the SARS-CoV-2 virus in the NCBI database as an example.

[0052] (1) In vitro amplification of viral genome fragments and in vitro splicing

[0053] The virus-encoding N gene was completely deleted and replaced with cDNA encoding GFP. At the same time, a T7 promoter sequence was placed upstream of the 5'UTR of the virus for in vitro transcription to initiate viral genome transcription. The entire viral genome is 29k long, divided into five segments A, B, C, D, and E, which were chemically synthesized by GenScript Biotechnology Company and cloned into the pCC1 vector ( figure 1 A, the sequence of fragments A-E is shown in SEQ ID NO: 1-5), wherein the gene encoding N protein in fragment E is replaced by the gene encoding GFP. Using the pCC1 vector cloned with fragments A-E as templates, use the following primers for PCR amplification and introduce IIS ty...

Embodiment 2

[0108] Experiments in which a cell line overexpressing the N protein of SARS-CoV was used to generate a recombinant SARS-CoV-2 virion whose genome lacked the gene encoding its own N protein.

[0109] This example shows the construction of a Caco-2 cell line that stably expresses the N protein of SARS-CoV (the sequence information is derived from the SARS-CoV Urbani strain, gene ID: MK062182.1), named Caco-2-N (SARS1 ). This cell line can also be used to support the packaging of SARS-CoV-2GFP virus whose genome lacks the N gene, suggesting that the function of the N protein in SARS-related viruses is very conserved. It also suggests that the scheme of Example 1 also has applicability in SARS-CoV virus.

[0110] (1) Construction of lentiviral expression vector expressing SARS-CoV N protein

[0111] The SARS-CoV virus N gene connected with the coding sequence of the FLAG tag was cloned into the lentiviral vector pLVX-IRES-mCherry, which was named pLVX-SARS-CoV N-IRES-mCherry. ...

Embodiment 3

[0115] Embodiment 3: drug evaluation experiment

[0116] This SARS-CoV-2 cell culture model can be used for rapid and efficient evaluation of antiviral regimens. This example evaluates the inhibitory effects of type I interferon IFN-β, remdesivir, ritonavir and lopinavir on viruses.

[0117] (1) Antiviral drugs:

[0118] The antiviral drugs selected in this example are type I interferon IFN-β (Sino Bioligical, 10704-HNAS-5); remdesivir (MedChemexpress, HY-104077); lopinavir (biochempartner, BCP01395) and Tonavir (biochempartner, BCP03777).

[0119] (2) Antiviral drug effect evaluation experiment:

[0120] Antiviral effect experiment of type I interferon IFN-β: the day before the experiment, Caco-2-N cells were mixed with 5×10 cells per well. 4 Cells were plated in 24-well culture plates containing DMEM medium. The next day, discard the culture medium in the 24-well culture plate, and dilute IFN-β with DMEM culture medium to image 3 The corresponding concentrations shown...

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Abstract

The present invention relates to recombinant coronavirus and application thereof to preparation of vaccines. A genome of the recombinant coronavirus has deficiency in a gene encoding structural protein, which results in that the recombinant virus produced solely from the genome of the recombinant virus lacks the structural protein. The invention also relates to a preparation method of the recombinant coronavirus. The preparation method comprises the following steps of (1) carrying out in-vitro amplification on virus genome segments and carrying out in-vitro splicing; (2) performing in-vitro transcription to prepare genomic RNA of the recombinant virus, wherein the genomic RNA has a mutation in the gene encoding the structural protein, which results in that the recombinant virus produced solely from the genome of the recombinant virus lacks the structural protein; (3) preparing a packaging cell line, wherein the packaging cell line stably expresses the structural protein which is lacked by the recombinant virus; and (4) transfecting the genome RNA of the recombinant virus into the packaging cell line to generate the recombinant virus. The invention also relates to the packaging cell line for preparing the recombinant coronavirus.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a coronavirus cell model based on genetic complementation of viral structural proteins. More specifically, the present invention relates to a novel coronavirus (SARS-CoV-2) cell model based on the genetic complementation of viral capsid proteins. Background technique [0002] The novel coronavirus (SARS-CoV-2) belongs to the Coronaviridae family, the Orthocoronavirinae subfamily, and the Betacoronavirus genus. The Betacoronavirus genus also includes SARS-CoV causing severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) and MERS-CoV causing Middle East respiratory syndrome (Middle East respiratory syndrome coronavirus, MERS). [0003] The new coronavirus (SARS-CoV-2) is a positive-sense single-stranded RNA virus with a genome length of about 30kb, with non-coding regions at both ends, and non-structural protein coding regions and structural protein coding regions ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/50C12N15/65C12N15/867C12N5/10A61K39/215A61P31/14C12R1/93
CPCC12N7/00C07K14/005C12N15/86C12N15/65A61K39/12A61P31/14C12N2770/20021C12N2770/20022C12N2770/20034C12N2770/20052C12N2740/15043
Inventor 丁强鞠晓辉
Owner TSINGHUA UNIV
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