Studies On Norovirus MRNA Vaccines

Objective: Two in vitro transcription systems for norovirus m RNA candidate vaccines were designed and constructed.By analyzing and comparing the expression differences,the best in vitro transcription system was screened out,which laid a theoretical and experimental foundation for the development of Norovirus m RNA vaccine in the later stage.Methods:(1)The EGFP reporter gene and No V-VP1 gene were inserted into the designed in vitro transcription system to construct the recombinant plasmid.(2)Preparation of m RNA by in vitro transcription cap purification using linearized DNA as a template.(3)m RNA transfection in vitro.(4)Functional verification of the two in vitro transcription systems was performed by microscopic fluorescence observation,enzyme-linked immunosorbent assay,indirect immunofluorescence assay,and Western blotting.Results:(1)The m RNA samples with good quality were successfully prepared by in vitro transcription cap purification.(2)Green fluorescence was observed under the excitation light of the microscope after transfection of the two groups of m RNA samples with EGFP reporter gene,and the fluorescence of m RNA-W1 group was stronger.(3)Two groups of m RNA samples inserted with GII.4 No V-VP1 target antigen were transfected into cells,and the ELISA results were positive.IFA microscopic observation showed obvious fluorescence;western Blot has obvious specific bands;the intracellular No V-VP1 protein was successfully expressed,and the m RNA-W1 group had better translation efficiency,protein expression and expression duration.Conclusion: Two in vitro transcription systems of Norovirus m RNA vaccine were successfully constructed,and both systems could express GII.4 No V-VP1 target protein.The expression effect of m RNA-W1 system was better than that of m RNA-W2 system.Finally,the m RNA-W1 in vitro transcription system was selected for the follow-up study of norovirus m RNA vaccine to help the prevention and control of norovirus in China.