Delta Variant (B.1.617.2) Spike Pseudotyping Vector

Delta (B.1.617.2) Spike Pseudotyping vector

ABOUT

For lentiviral particle pseudotyping with the SARS-CoV-2 Spike (B.1.617.2 - Indian origin) 

pLV-SpikeV8 has been specifically designed for the pseudotyping of lentiviral particles with the SARS-CoV-2 Spike (S) protein when co-transfected with plasmids encoding a reporter and accessory proteins (not provided). pLV-SpikeV8 encodes the full-length Spike sequence from the Delta variant (B.1.617.2), and for optimal expression, it is codon-optimized and the C‑terminal ER-retention signal has been removed [1, 2].

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Gene Description

This plasmid encodes the Spike protein from the SARS-CoV-2 Delta variant, first reported in October 2020 in India. This variant is classified as a member of Clade 21A/ B.1.617.2 lineage (Nextstrain/Pango lineage classification). It is characterized by the presence of mutations within the Spike coding region, of which, several are of concern [3,4].

  • S1 domain: T19R, T95I, G142D, E156G, deletion (Δ)F157-R158, D614G, P681R
  • RBD: L452R, T478K
  • S2 domain: D950N

Learn more about SARS-CoV-2 variants

 

General Plasmid Description

This plasmid features a potent mammalian expression cassette composed of the ubiquitous human-(h)CMV composite promoter, a rabbit β-globin intron directly upstream of the spike gene, and a rabbit β-globin polyadenylation (pAn) signal. The spike coding sequence includes the SARS-CoV-2 signal sequence and the S1/S2 furin cleavage site [5]. These plasmids are selectable with ampicillin in E. coli.

 

Applications

  • Generation of Spike pseudotyped lentiviral particles upon co-transfection with reporter and accessory protein plasmids (not provided).
  • Screening of SARS-CoV-2 inhibitors including small molecules, monoclonal antibodies, or convalescent plasma.

 

References:

1. Johnson, M.C. et al. 2020. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. J Virol 94.
2. Ou, X. et al. 2020. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 11, 1620.
3. Davis, C. et al. 2021. Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination. medRxiv doi:10.1101/2021.06.23.21259327.
4. Centers for Disease Control and Prevention. SARS-CoV-2 Variant Classifications and Definitions. Retrieved 07 July 2021.
5. Coutard, B. et al. 2020. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 176, 104742.

All products are for internal research use only, and not for human or veterinary use.

SPECIFICATIONS

Specifications

Accession number

EPI_ISL_2356230

ORF size
3759 bp
Plasmid backbone
pLV
Gene promoter
hCMV
Purification
Ion-exchange chromatography
Reconstitution buffer
Sterile water
Tested applications

Generation of Spike pseudotyped lentiviral particles, Screening of SARS-CoV-2 inhibitors

Quality control

Plasmid construct is confirmed by restriction analysis and full-length open reading frame (ORF) sequencing. After purification by ion exchange chromatography, predominant supercoiled conformation is verified by electrophoresis.

CONTENTS

Contents

  • Product: 
    pLV-SpikeV8
  • Cat code: 
    plv-spike-v8
  • Quantity: 
    20 µg

Shipping & Storage

  • Shipping method:  Room temperature

    • Storage:

    • -20°C

Details

Spike-pseudotyping of lentiviral particles

InvivoGen's pLV-Spike plasmid collection has been designed for pseudotyping lentiviral particles with the SARS-CoV-2 Spike (S) protein. The S protein a structural glycoprotein expressed on the surface of SARS‑CoV-2. It mediates membrane fusion and viral entry into target cells upon binding to the host receptor ACE2 and its cleavage by cellular proteases such as TMPRSS2 [1]. Notably, the C‑terminal cytoplasmic tail of the S protein encodes a presumptive endoplasmic reticulum (ER)‑retention motif (KxHxx), which has previously been shown to enable the accumulation of SARS‑CoV S proteins at the ER‑Golgi intermediate compartment (ERGIC) and facilitate their incorporation into new virions [2]. The removal of this motif (d19) has been shown to increase the expression of the spike protein in pseudovirions [3,4].

Pseudotyped particle production involves the co-transfection of 293T cells with a reporter protein vector (e.g. GFP), one or several plasmids encoding the necessary lentiviral proteins, and the pseudotyping pLV-Spike plasmids. The transfected cells produce SARS‑CoV‑2 Spike (S)‑pseudotyped lentiviral particles, which can then be used to infect permissive cells, such as ACE2‑expressing HEK293-derived cells and ACE2‑TMPRSS2‑expressing A549-derived cells.

 

References

1. Hoffmann M. et al. 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 181:1-16.
2. Ujike, M. et al. 2016. The contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of S proteins and incorporation of S protein into virus-like particles. J Gen Virol 97, 1853-1864.
3. Johnson, M.C. et al. 2020. Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein. J Virol. 94(21):e01062-20
4. Ou, X. et al. 2020. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 11, 1620.

DOCUMENTS

Documents

pLV-SpikeV8

Technical Data Sheet

Plasmid Sequence

Safety Data Sheet

Certificate of analysis

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