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Updated: Jun 15, 2025

Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring
Published on: April 4, 2025
Optimization of the 5' untranslated region of mRNA vaccines
Qi Ma1, Xiaoguang Zhang1, Jing Yang1
1National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Yingxin Street #100, Xicheng District, Beijing, 100052, China.
Abstract:
To investigate the impact of different 5' untranslated regions (UTRs) on mRNA vaccine translation efficiency, five dual-reporter gene expression plasmids with different 5'UTRs were constructed. The corresponding mRNA transcripts were transcribed and capped in vitro. By comparing the expression levels of reporter genes with different 5'UTRs, we identified the 5'UTR associated with the highest expression level. Subsequently, HIVgp145 mRNA vaccines containing various 5'UTRs were constructed and verified. The results demonstrated that mRNA 3 (β-globin 5'UTR) displayed the greatest number of green fluorescence-positive cells and the highest luciferase fluorescence intensity in the reporter gene expression system. Further, among the HIVgp145 mRNA vaccines with different 5'UTRs, mRNA 7 (β-globin 5'UTR) exhibited the highest level of expression. These findings indicate that it is feasible to use the 5'UTR of β-globin in an mRNA vaccine, laying the foundation for animal immunogenicity testing.
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