D614 Varient Spike Pseudotyping Vector

Original Spike (D614) Pseudotyping vector

ABOUT

For lentiviral particle pseudotyping with the SARS-CoV-2 Spike (D614)

pLV-Spike 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-Spike encodes the full-length Spike sequence from the original isolate first isolated in Wuhan, 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 original SARS-CoV-2 isolate, first reported in Wuhan in December 2019 [3]. This sequence is characterized by the presence of D614 and is classified as the root of the pandemic in Clade 19A/ Lineage A (Nextstrain/Pango lineage classification). 

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 [4]. 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. Zhou, P. et al. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273.
4. 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 research use only, and not for human or veterinary use.

Wuhan-Hu-1 Spike (D614) Spike pseudotyping vector
Wuhan-Hu-1 Spike (D614) Spike pseudotyping vector

SPECIFICATIONS

Specifications

Accession sequence

NC_045512.2

ORF size
3765 bp
Plasmid backbone
pLV
Subclone start
AgeI
Subclone end
NheI
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-Spike
  • Cat code: 
    plv-spike
  • Quantity: 
    20 µg

Shipping & Storage

  • Shipping method:  Room temperature

    • Storage:

    • -20°C
    Stability: Resuspended DNA should be stored at -20°C and is stable for at least 1 year.

    Caution:

    • Avoid repeated freeze-thaw cycles

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-Spike

Technical Data Sheet

Plasmid Sequence

Safety Data Sheet

Certificate of analysis

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