Application of 12C6 Heavy Ion-Irradiated BHK-21 Cells in Production of Foot-and-Mouth Disease Vaccine

Figures

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  Figure 1. (a) BHK-21 cells were induced with 12C6 heavy ions at doses of 0 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, and 25 Gy. Cell viability was assessed via trypan blue staining and detected via Count STAR (Countstar Rigel S2, RY074B2001), and mortality rate was calculated. Data represent means and standard errors from six independent experiments. (b) After BHK-21 cells were induced with different doses, foot- and -mouth disease virus (FMDV) was used to infect cells at infection multiplicity (MOI) of 0.1 for 16 h. (c) After researchers exposed BHK-21 cells to irradiation doses of 5 Gy, 10 Gy, and 15 Gy, mutant cells obtained through monoclonal screening were infected with FMDV at multiplicity of infection (MOI) of 0.1 for 16 h, resulting in alterations in antigen content, specifically in the 146S component. Experiments were conducted in triplicate as biological replicates. Data are presented as means ± SDs from three independent experiments and were analyzed via Student’s two-tailed unpaired t-tests. *, p < 0.05; ***, p < 0.001. The orange bar graph represents the 146s content after FMDV infection in the mutant cell group, and the white bar graph represents the 146s content after FMDV infection in…

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  Figure 2. (a) Shows expression level of FMDV genes in mutant BHK-7 and control BHK-21 cells infected with FMDV at multiplicity of infection (MOI) of 0.1 for 16 h. (b) Shows change in 146S antigen content in mutant BHK-7 and control BHK-21 cells infected with FMDV at multiplicity of infection (MOI) of 0.1 for 16 h. (c) Shows change in TCID50 after mutant BHK-7 and control BHK-21 cells are infected with FMDV. (d) Shows growth curves of mutant BHK-7 and control BHK-21 cells. Experimental data are all from three biological replicates. Data are presented as means ± SDs from three independent experiments and were analyzed via Student’s two-tailed unpaired t-tests.; ****, p < 0.0001.

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  Figure 3. (a) BHK-7-FC suspension cells were stained with trypan blue for viability assessment and bright-field imaging. (b) BHK-21 suspension cells were stained with trypan blue for viability assessment and bright-field imaging. (c) Proliferation curve of BHK-7-FC cells in suspension culture is presented. (d) Short-term viability of BHK-7-FC cells in suspension culture over time was evaluated. Data are presented as means ± SDs from three independent experiments. Red circles represent clumped cells.

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  Figure 4. (a) Temporal variations in GLN content following FMDV infection in BHK-7-FC suspension cultures. (b) Changes in NH4+ levels at various time points post-FMDV infection in BHK-7-FC suspension cultures. (c) Temporal changes in LAC content following FMDV infection in BHK-7-FC suspension cultures. (d) Variations in GLUC content over time after FMDV infection in BHK-7-FC suspension cultures. (e) Effect of supplementing different concentrations of GLN on 146S antigen content 16 h post-FMDV infection; (f) Impact of adding 1 mmol/L GLN to both BHK-7-FC and control BHK-21 cells on 146S antigen content 16 h post-FMDV infection. All experiments were conducted with three biological replicates. Data are presented as means ± SDs from three independent experiments and were analyzed via Student’s two-tailed unpaired t-tests. *, p < 0.05; **, Black and white dots represent the scatter points on the column, indicating the specific data of each sample within the group.

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  Figure 5. (a) The BHK-7-FC cell line was passaged for 10 successive generations. Following FMDV infection, the 146S antigen content was assessed every three generations compared with the control BHK-21 cells. Additionally, the replication stability of the mutant BHK-7 cells after suspension culture adaptation was evaluated. (b) BHK-7-FC cells were passaged for 10 consecutive generations, and their TCID50 was measured every three generations after FMDV infection, in comparison with control BHK-21 cells. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student’s two-tailed unpaired t-tests. ***, p < 0.001; ****, p < 0.0001. T3, represents the third passage of cells, T6, represents the sixth passage of cells, and T9 represents the ninth passage of cells.