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Recombinant VSV virus for expressing SARS-CoV-2 spike protein (S) or variant thereof, and construction and application thereof

A spike protein, sars-cov-2 technology, applied in the direction of antisense single-stranded RNA virus, positive-sense single-stranded RNA virus, virus, etc., can solve the limitation of the use and promotion of neutralizing antibody detection methods, the slow replication speed of coronavirus And other issues

Pending Publication Date: 2021-09-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, neutralizing antibody-based detection methods also have some bottlenecks, including: (1) neutralizing antibody detection usually requires the use of the original live virus, for highly pathogenic viruses like CoV-2, this means that it is necessary to Only laboratories can carry out relevant tests; (2) The judgment of the results depends on the obvious lesions (CPE) of the cells, which has a certain degree of subjectivity; (3) The replication speed of coronaviruses is slow, and the judgment time of CPE usually takes 72 hours
These problems have greatly limited the use and promotion of neutralizing antibody detection methods

Method used

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  • Recombinant VSV virus for expressing SARS-CoV-2 spike protein (S) or variant thereof, and construction and application thereof
  • Recombinant VSV virus for expressing SARS-CoV-2 spike protein (S) or variant thereof, and construction and application thereof
  • Recombinant VSV virus for expressing SARS-CoV-2 spike protein (S) or variant thereof, and construction and application thereof

Examples

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Effect test

preparation Embodiment 1

[0056] Preparation Example 1: Optimizing the length of the intracellular segment (cytoplasmic tail, CT) of the SARS-CoV-2S protein so that it can be efficiently embedded in the VSV capsule

[0057] The SARS-CoV-2 virus S gene required in this example will be synthesized according to the Wuhan-Hu-1 virus strain sequence (GenBank accession no. MN908947) published on NCBI. The structure of coronavirus S protein is as follows figure 1, it is a type I transmembrane protein, including N-terminal signal peptide, extracellular domain (Ecotdomain, ET), transmembrane domain (Transmembrane domain, TM) and intracellular domain (cytoplasmic tail, CT) and other four parts. There is a KxHxx motif structure (AA1269-1273) at the carboxyl terminus of the SARS-CoV-2S protein. In this example, the carboxy-terminal amino acids of the S protein were knocked out one by one, and 1 to 26 amino acids in the intracellular segment were knocked out, including the removal of the KxHxx motif structure, and...

preparation Embodiment 2

[0063] Preparation Example 2: pVSV ΔG -S ΔCT Recombinant plasmid construction

[0064] 1. Construct pVSV IND Plasmid, the plasmid clone has the full-length genome sequence of VSV Indiana strain;

[0065] 2. Put S ΔCT The gene was amplified by high-fidelity PCR, and its 5' and 3' ends contained MluI and XhoI restriction sites, respectively, and cloned into pVSV after double digestion IND In the plasmid, make S ΔCT The G gene in VSV is replaced by the gene, and the recombinant plasmid pVSV is obtained ΔG -S ΔCT ,like figure 2 shown.

preparation Embodiment 3

[0066] Preparation Example 3: pVSV ΔG -S recombinant plasmid construction

[0067] 1. Construct pVSV IND Plasmid, the plasmid clone has the full-length genome sequence of VSV Indiana strain;

[0068] 2. The full length of the S gene was amplified by high-fidelity PCR, and its 5' and 3' ends contained MluI and XhoI restriction sites respectively, and cloned into pVSV after double digestion IND In the plasmid, the S gene is replaced by the G gene in VSV to obtain the recombinant plasmid pVSV ΔG -S.

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Abstract

The invention provides a recombinant VSV virus for expressing an SARS-CoV-2 spike protein (S) or a variant thereof, and construction and application thereof. The construction of the recombinant VSV virus comprises any one of the following methods: constructing a pseudotyped VSV virus through an S protein of the SARS-CoV-2 or a variant S delta CT protein of the SARS-CoV-2; and adopting an M three-site mutated VSV virus, an M and G protein mutated VSV virus or a structural protein sequence rearranged VSV virus as a vector to construct a recombinant VSV virus expressing the S protein of the SARS-CoV-2 or the variant S delta CT thereof. The constructed recombinant VSV virus is carried out aiming at the S protein of the SARS-CoV-2 virus, so that the live virus is not involved, and the biological safety problem cannot be generated. The constructed recombinant VSV virus can be used for developing vaccines and detecting SARS-CoV-2 virus specific neutralizing antibodies in human bodies and animal bodies.

Description

technical field [0001] The invention relates to the technical field of novel coronavirus research, in particular to a recombinant vesicular stomatitis virus (VSV) expressing the spike protein (S) of SARS-CoV-2 or a variant thereof and its construction and application. Background technique [0002] SARS-CoV-2 (hereinafter referred to as CoV-2) is the seventh known human coronavirus. According to serotype and genome characteristics, Coronaviridae is divided into four genera: α, β, γ and δ. CoV-2 belongs to the betacoronavirus genus, which also includes Middle East respiratory syndrome-associated coronavirus (MERS-CoV) and severe respiratory syndrome-associated coronavirus (SARS-CoV). Phylogenetic analysis shows that the similarity between the new coronavirus and SARS virus is higher than that of MERS virus. [0003] The CoV-2 genome is a single-stranded positive-strand RNA with a length of about 29.8Kb, and its 5' end is the open reading frame (Open reading frame, ORF) 1ab, ...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N7/01G01N33/68G01N33/569A61K39/215A61P31/14
CPCC12N15/86C07K14/005G01N33/68G01N33/56983A61K39/12A61P31/14C12N2770/20022C12N2760/20243G01N2333/165C12N2770/20034A61K2039/5256
Inventor 孙涛方心葵
Owner SHANGHAI JIAO TONG UNIV
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