Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/4813
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dc.contributor.authorDahiya, Surbhi-
dc.contributor.authorSingh, Sudhakar-
dc.contributor.authorSehrawat, Sharvan-
dc.date.accessioned2023-08-17T17:48:57Z-
dc.date.available2023-08-17T17:48:57Z-
dc.date.issued2022-
dc.identifier.citationSTAR Protocols, 4(1), 1-14.en_US
dc.identifier.urihttps://doi.org/10.1016/j.xpro.2022.101977-
dc.identifier.urihttp://hdl.handle.net/123456789/4813-
dc.descriptionOnly IISERM authors are available in the recorden_US
dc.description.abstractThis protocol provides detailed methodology used in the published article; Robust anti-SARS-CoV-2 single domain antibodies cross neutralize multiple viruses.1 The protocol describes a strategy for the intracellular expression of sdAbs targeting the polybasic cleavage site (anti-CSP sdAb) of the viral entry mediator, SARS-CoV-2 S protein. We also analyze the intracellular fate of the protein in the anti-CSP intrabody (IB)-expressing cells. For generating an anti-CSP IB construct, the sequence of the selected sdAb specific to peptide (NSPRRAR/SVAS) which encompasses the polybasic cleavage site (CSP) of SARS-CoV-2 S protein was cloned into a pLenti-GFP vector (a lentivirus-based mammalian expression vector) downstream to CMV promoter.2 The cloning scheme used is: 50 LTR-CMV promoter-VHH - c-Myc-tag - e-GFP -30 -LTR. The anti-CSP sdAb sequence was amplified using the forward primer, VHH-FR1-pLenti (containing XbaI restriction site), and reverse primer, FR4-c-Myc pLenti, that encoded c-Myc tag at 30 end of VHH with a BamHI site) (Figure 1). To generate the lentivirus-based pseudoviruses expressing surface SARS-CoV-2 S protein, HEK293T cells were transfected with plasmids such as a reporter construct, pCMVR8.74, SARS-CoV-2 S construct, tat and rev as well as pMD2.G.3 The plasmids required for producing LV(CoV-2 S) pseudoviruses were prepared and the purity was confirmed before beginning with the experiments.en_US
dc.language.isoen_USen_US
dc.publisherElsevieren_US
dc.subjectSARS-CoV-2 S pseudovirusesen_US
dc.subjectHEK293T cellsen_US
dc.subjectvirus-specific intrabodiesen_US
dc.titleProtocol for investigating the biogenesis of SARS-CoV-2 S pseudoviruses in HEK293T cells transduced to express the virus-specific intrabodiesen_US
dc.typeArticleen_US
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