mRNA was transcribed in vitro from a linearized DNA template. This template encompassed LUC (Luciferase, GenBank: Q27758), the wildtype RBD from the Spike (S) protein of SARS-CoV-2 (GenBank: P0DTC2), and signal sequences of origin (GenBank: P0DTC2), IL-6 (GenBank: P08505), and tPA (GenBank: P11214). The transcription process employed T7 RNA polymerase (Vazyme Biotech Co., Ltd).
T7 rna polymerase
Manufactured by Vazyme
Sourced in China
T7 RNA polymerase is an enzyme responsible for the transcription of DNA into RNA. It specifically recognizes and binds to the T7 promoter sequence, allowing it to initiate the synthesis of RNA molecules from a DNA template.
Automatically generated - may contain errors
3 protocols using t7 rna polymerase
1
SARS-CoV-2 Spike RBD mRNA Production
2
In Vitro Transcription and Purification of mRNA
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The recombinant plasmids encoding ALAB and four single genes (Sin1 to Sin4) were extracted and linearized by restriction endonuclease BspQ I cleavage followed by plasmid recovery and purification. In vitro transcription reaction (IVT in 50 μL) was performed at 37 °C for 3 h after vortexing of the mixture containing linearized plasmid template, ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM), Cap analogue (100 mM, Vazyme, Nanjing, China), T7 RNA polymerase (200 U/μL, Vazyme), 10X T7 Reaction Solution (Vazyme), RNase enzyme inhibitor (40 U/μL, Vazyme), pyrophosphatase (0.1 U/μL, Vazyme), and RNase Free H2O (Invitrogen, Carlsbad, CA, USA, 10977015). After the IVT, 170 μL of Rnase-free H2O, 5 μL of DNaseI enzyme (NEB, Beverly, MA, USA, M0303L), and 25 μL of 10× DNaseI enzyme reaction solution (NEB, M0303L) were added to the mRNA and incubated at 37 °C for 20 min. Purified mRNA was obtained using OligoDT beads (Vazyme, N401). The mRNA integrity and purity were analyzed using Agarose Gel (Bio-Rad, Hercules, CA, USA) and HPLC-SEC (Thermo Fisher Scientific, Waltham, MA, USA).
3
Synthesis of SARS-CoV-2 Spike Protein Variants
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The full length spike with the KEN445 mutants (K417N-E484K-N501Y, KEN445) or the spike with the Omicron BA.5 mutants were synthesized respectively (Genscript, China). Both plasmids contained HBB 5'UTR, a CDS region encoding amino acid residues 1 through 1205 of the SARS-CoV-2 spike protein with the following mutations: a "GSAS" substitutions at the Furin cleavage site (residues 682–685), 6 proline substitutions at residues 817, 892, 899, 942, 986 and 987, and a C-terminal T4 fibritin trimerization motif, two tandem HBA2 3’UTR, 120 polyA tail. The sequences were cloned into a pUC57-kan vector. The mRNA encoding KEN445 mutants or BA.5 specific spike antigens were manufactured via in vitro transcription (IVT) with T7 RNA polymerase (Vazyme, China). Unmodified 5-triphosphate was completely replaced with N-1-methylpseudouridine-5-triphosphate (Synthgene, China). After IVT, the Cap-1 structure was added to the 5' terminus using vaccinia capping system and 2'-O- methyltransferase (Vazyme). The mRNA was purified with LiCl precipitation, resuspended in 1 mM sterile-filtered sodium acetate (pH 6.5), and kept frozen at − 80 °C until use.
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