Spike(SARS-CoV-2) Pseudotyped Lentivirus (Luc-eGFP Dual Reporter)
The pandemic coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As the first step of viral replication, the virus attaches to the host cell surface before entering the cell. The viral Spike protein recognizes and attaches to the Angiotensin-Converting Enzyme 2 (ACE2) receptor found on the surface of type I and II pneumocytes, endothelial cells, and ciliated bronchial epithelial cells. Drugs targeting the interaction between the Spike protein of SARS-CoV-2 and ACE2 may offer protection against the viral infection.
The SARS-CoV-2 Spike Pseudotyped Lentivirus (Luc-eGFP dual reporter) were produced by replacing the VSV-G fusion glycoprotein with SARS-CoV-2 Spike protein (Genbank Accession #QHD43416.1) as a surrogate viral envelope protein. These pseudovirions also contain a firefly luciferase and eGFP cassette (Luc-P2A-eGFP) driven by a CMV promoter (Figure 1). The luciferase and eGFP are coexpressed under the CMV promoter in the transduced cells. Therefore, the Spike-mediated entry into the target cell can be conveniently measured via luciferase reporter activity or eGFP expression. The SARS-CoV-2 Spike pseudotyped lentivirus can be used in a cellular assay to measure the activity of neutralizing antibody against SARS-CoV-2.
Spike interacts with host cells by binding to membrane receptor ACE2 (angiotensin converting enzyme 2). Based on experiments performed by scientists at BPS Bioscience, we know that the wild-type SARS-CoV-2 spike pseudotyped lentiviruses transduce the following cells with great efficiency: ACE2-HEK293 cells (BPS Bioscience #79951), ACE2-CHO cells (BPS Bioscience #79959), ACE2-HeLa cells (BPS Bioscience #79958). They also efficiently transduce TMPRSS2-Vero E6 cells (BPS Bioscience #78081), which express high endogenous levels of ACE2 and were stably transfected with human serine protease TMPRSS2 required for the priming of Spike and fusion of the virion with the plasma membrane. By contrast, it has been shown by others that SARS-CoV-2 spike pseudotyped lentiviruses do not transduce parental Calu3 and Vero E6 cells very well [Neerukonda et al. 2021, PlosOne PMID: 33690649; Tandon et al. 2020, Scientific Reports PMID: 33154514; Condor Capcha et al. 2021, Front. Cardiovasc. Med. PMID: 33521067; Pisil et al. 2021, Pathogens PMID: 33540924].
As recommended in our protocol, 5 µl of virus/well in a 96-well plate provides a sufficient signal-to-noise ratio to perform inhibition studies. The amount of virus added to the cells can also be scaled down according to the user’s need.
Figure 1. Schematic of the Luciferase-P2A-eGFP Reporter in SARS-CoV-2 Spike Pseudovirion
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| Name | Ordering Information |
| HEK293 growth medium or use of Thaw Medium 1 | BPS Bioscience, #60187 |
| ACE2-HEK293 Recombinant Cell Line | BPS Biocience, #79951 |
| Bald Lentiviral Pseudoviron (Luc-eGFP dual reporter) | BPS Bioscience, #79988 |
| Neutralizing Anti-SARS-CoV-2 Spike antibody | BPS Bioscience, #100793 |
| 96-well tissue culture treated, white clear-bottom assay plate | Corning, #3610 |
| ONE-Step™ luciferase assay system | BPS Bioscience, #60690 |
The functional titer of SARS-CoV-2 Spike Pseudotyped Lentivirus (~1-2 x 105 TU/ml) is significantly lower than VSV-G pseudotyped lentivirus (>107 TU/ml), although the amount of lenti particles are similar (>109 LP/ml). The exact titer is provided with each shipment.
The lentiviruses were produced from HEK293T cells in medium containing 90% DMEM + 10% FBS.
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