Construction and application of pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or variant Sdelta21 of SARS-CoV-2 S protein

A spike protein, sars-cov-2 technology, applied in antisense single-stranded RNA virus, positive-sense single-stranded RNA virus, applications, etc., can solve the slow replication speed of coronavirus, limit the use and promotion of neutralizing antibody detection methods and other problems, to achieve the effect of fast replication and easy in vitro amplification

Pending Publication Date: 2021-12-21
SHANGHAI JIAO TONG UNIV
View PDF6 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or variant Sdelta21 of SARS-CoV-2 S protein
  • Construction and application of pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or variant Sdelta21 of SARS-CoV-2 S protein
  • Construction and application of pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or variant Sdelta21 of SARS-CoV-2 S protein

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0051] 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

[0052] The SARS-CoV-2 virus S gene required by this embodiment will carry out the whole gene synthesis according to the virus strain sequence (GenBankaccession no.MN908947) announced on the NCBI, and the codon of the synthetic gene is optimized according to the human cell codon (SEQID NO.1 ), the sequence was published for the first time, and it has been proved by experiments that the protein can be expressed at a higher level compared with the spike protein S gene sequence of the original new coronavirus strain, which is shown in the fact that we successfully used this sequence to construct Novel COVID-19 vaccine based on recombinant vesicular stomatitis virus. The structure of coronavirus S protein is as follows figure 1 , it is a type I transmembrane protein, including N-terminal signa...

preparation Embodiment 2

[0062] Preparation Example 2: pVSV ΔG -S, pVSV ΔG -S Δ21 Recombinant plasmid construction

[0063] 1. Construct pVSV IND Plasmid, the plasmid clone has the full-length genome sequence of VSV Indiana strain;

[0064] 2. Perform high-fidelity PCR amplification of the S or SΔ21 gene, whose 5' and 3' ends contain MluI and XhoI restriction sites respectively, and clone into pVSV after double digestion IND In the plasmid, make S Δ21 The G gene in VSV is replaced by the gene, and the recombinant plasmid pVSV is obtained ΔG -S or pVSV ΔG -SΔ21, as figure 2 shown.

preparation Embodiment 3

[0065] Preparation Example 3: pVSV MTΔG -S, pVSV MTΔG -SΔ21 recombinant plasmid construction

[0066] 1. Construct pVSV MT Plasmid, its plasmid clone has the genome sequence after the M three site mutation of VSV Indiana strain;

[0067] 2. Perform high-fidelity PCR amplification of the S or SΔ21 gene, whose 5' and 3' ends contain MluI and XhoI restriction sites respectively, and clone into pVSV after double digestion MT In the plasmid, the recombinant plasmid pVSV was obtained MTΔG -S or pVSV MTΔG -SΔ21, as figure 2 shown.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention provides construction and application of a pseudotyped VSV (vesicular stomatitis virus) for expressing SARS-CoV-2 spike (S) protein or a variant Sdelta21 of the SARS-CoV-2 S protein. The construction comprises the following steps: knocking out a G protein gene of the VSV responsible for identifying a host cell receptor, and constructing the pseudotyped VSV through the S protein of SARS-CoV-2 or the variant Sdelta21 protein of the SARS-CoV-2; and Sdelta21 is an S protein variant with 21 amino acids deleted in an intracellular region. The constructed recombinant VSV is an S protein variant obtained by correspondingly deleting the S protein of the SARS-CoV-2 virus and the carboxyl terminal of the S protein, so that the recombinant VSV does not relate to a live virus and does not generate a biological safety problem; and besides the construction of the VSV taking wild type M protein as a skeleton, a brand new thought is also provided, that is, the recombinant VSV constructed by using the M protein can be used for the development of vaccines and the detection of SARS-CoV-2 virus specific neutralizing antibodies in human and animal bodies.

Description

technical field [0001] The invention relates to the technical field of novel coronavirus research, relates to a pseudotyped VSV virus expressing SARS-CoV-2 spike protein (S) or its variants and its construction and application; Recombinant vesicular stomatitis virus (VSV) with deletion of the spike protein (S) of 2 or its carboxyl terminus and its construction and application. Background technique [0002] 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 ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C07K14/165C12N7/01C12N15/86A61K39/12A61P31/14G01N33/68
CPCC07K14/005C12N7/00C12N15/86A61K39/12A61P31/14G01N33/6854C12N2770/20022C12N2770/20034C12N2760/20221C12N2760/20234C12N2760/20243C12N2800/22
Inventor 孙涛张宝红柯勇
Owner SHANGHAI JIAO TONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap