Table 1

A pair of EGFP primers"

Fig. 1

Schematic diagram of mRNA transcription vectors in vitro A. mRNA sequences designed according to the mRNA vaccine platform; B. Constructed recombinant plasmid containing mRNA transcription elements"

Fig. 2

CAI index, GC content, secondary structure, and bond energy magnitude before and after FIPV N sequence optimization"

Fig. 3

Construction and identification of recombinant plasmids A. In vitro amplification of the EGFP gene, purified agarose gel after double digestion (lane 1: in vitro amplification of the EGFP gene, lane 2: purification after double digestion of the EGFP gene); B. Agarose gel electropherogram of double digestion of the pBluescript Ⅱ KS(+)-FIPV N recombinant plasmid (lane 1), agarose gel electropherogram of gel-recovered mRNA transcription vector in vitro (lane 2); C. Extraction of recombinant plasmids and linearized agarose gel plots of single digestion (Lane 1: pBluscript lI KS(+)-EGFP plasmid; Lane 2: pBluscript Ⅰ KS(+)-FIPV N plasmid; Lane 3: Single enzyme digested pBluscript Ⅱ KS(+)-FIPV; Lane 4: Single enzyme cleaved pBluscript Il KS(+)-EGFP); D. In vitro transcribed EGFP-mRNA and FIPV N-mRNA agarose gel plots (lane 1:EGFP-mRNA; lane 2:FIPV N-mRNA)"

Fig. 4

Green fluorescent protein expression in 293T cells transfected with EGFP-mRNA transfected with Lipofectamine 3000"

Fig. 5

Expression of FIPV N protein in 293T cells among different trement groups M. Standard protein molecular weight; 1. Negative control group without transfection of FIPV N-mRNA; 2. Expression of FIPV N protein 12 hours after transfection of FIPV N-mRNA using Lipofectamine 3 000; 3. Expression of FIPV N protein 12 hours after transfection of FIPV N-mRNA using Lipofectamine 3 000"

Fig. 6

Expression and localization of FIPV N protein detected by indirect immunofluorescence assay in 293T cells transfected with FIPV N-mRNA"

Fig. 7

The immune response elicited by FIPV N-mRNA vaccine in mice after immunization A. Animal experimental immunology flow chart; B. Serum levels of FIPV N-specific antibodies in the grouped mice during immunization; C. TNF-α and IL-4 levels in the serum of the grouped mice during immunization; D. Elispot assay was used to detect the secretion level of IFN-γ factor in mouse splenocytes in each group. ** represents P < 0.01, with statistical difference, **** represents P < 0.000 1, which means significant statistical difference, ns indicates no statistical difference"