Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo

Chunxi Zeng; Xucheng Hou; Jingyue Yan; Chengxiang Zhang; Wenqing Li; Weiyu Zhao; Shi Du; Yizhou Dong

The sequences necessary for in vitro transcription of mRNA, including T7 promoter, 5' UTR, coding sequence, and 3' UTR, were cloned into pUC19 vector using repliQa HiFi Assembly Mix (QuantaBio) and transformed into 5-alpha Competent E. coli (NEB) by chemical transformation. The transformed E. coli was allowed to grow on LB broth (Miller) plate with agar and 100ug/mL carbenicillin (Teknova). Individual colonies were inoculated and outgrown in LB broth liquid medium (Miller) containing 100ug/mL carbenicillin overnight with vigorous shaking at 250 rpm. Plasmids were extracted using QIAprep Spin Miniprep Kit (Qiagen). Concentration was measured on a NanoDrop 2000 Spectrophotometer (Thermo). The region of interest in the plasmid from the T7 promoter to 3' UTR was confirmed by Sanger Sequencing. mRNA synthesis All mRNA transcripts were synthesized by in vitro transcription using a protocol modified from our previous publication 49 . Briefly, DNA templates were synthesized by PCR amplification of the corresponding plasmids using a forward primer and a reverse primer containing 120T at 5' end. The DNA templates were purified by QIAquick PCR Purification Kit (Qiagen) and examined by agarose gel electrophoresis. All mRNAs were synthesized by in vitro transcription with 100% substitution of UTP by pseudouridine-5'triphosphate (TriLink) using AmpliScribe T7-Flash Transcription Kit (Lucigen) following the manufacturer's instruction and purified by RNA Clean & Concentrator (Zymo). The capping of mRNA was conducted using Vaccinia Capping System (NEB) and Cap 2´-O-Methyltransferase (NEB), followed by another purification by RNA Clean & Concentrator (Zymo). After measurement of concentration by a NanoDrop 2000 Spectrophotometer (Thermo), all mRNAs were diluted to the desired concentration in 1× TE, aliquoted, and stored at -80°C for future use.