Differentially expressed genes.
After MTB infection, the gene expression in mice was significantly abnormal, there were 777 DE genes up-regulated and 1581 DE genes down-regulated. The gene expression variation between the TB model group and the normal control group or between each ag85a/b DNA treatment group and TB model group were analyzed and visualized by the scatter plot, volcano plot, and cluster plot (shown in Supplementary Figure 1). Many DE genes significantly up-regulated or down-regulated in the TB model group were recovered to varying degrees in eachag85a/b DNA vaccine treatment group. The total number of DE genes and abnormal DE gene recovered (ADEGR) in each group were shown in Fig. 2. The results showed that: (1) The gene expression was significantly abnormal in mice infected with MTB. Most DE genes with significantly up-regulated or down-regulated expression were recovered after treatment with 100μg, 200μg DNA IM, or 10-200μg DNA EP. (2) The number of DE genes up-regulated or down-regulated in the ag85a/b DNA IM group was positively correlated with the DNA injection dose. When the IM dose increased from 50μg to 100μg, both the number of up-regulated DE genes and the number of down-regulated DE genes increased greatly, while when the injection dose increased from 100μg to 200μg, the number of up-regulated and down-regulated DE genes increased slowdown. (3) The number of up-regulated DE genes in each dose of the DNA EP group was negatively correlated with the immune dose, but the downward trend was not obvious. There was no obvious correlation between the number of down-regulated DE genes and the dose of DNA EP. (4) The number of DE genes in the 10μg DNA EP group was equivalent to that in the 200μg DNA IM group.
The changes of the top 20 DE genes significantly up-regulated and down-regulated after MTB infection or after treatment withag85a/b DNA vaccine IM and EP are shown in Tables 2 and 3, respectively. These genes may be involved in the occurrence and development of TB, and reveal the new potential preventative and immunotherapeutic targets of the ag85a/b DNA vaccine for TB. The results show that: (1) The significantly up-regulated or down-regulated top 20 DE genes in the TB model group had no significant change in the 10μg DNA IM group and 50μg DNA IM group (P value ≥ 0.05) and were all recovered significantly in 100μg DNA IM group and 200μg DNA IM group (P value < 0.05). (2) The abnormally up-regulated or down-regulated DE genes in the TB model group were all recovered in the 4 ag85a/b DNA EP group. Furthermore, the Fold Change values of up-regulated or down-regulated DE genes in the TB model group were reversed in 4 DNA EP groups. (3) Most of the up-regulated DE genes in the TB model group were related to the digestion and absorption of nutrients or neuroendocrine (Table 2), for example, Iapp, Scg2, Amy2a5, Try5, Chga, Cpa1, Gcg, etc. Most of the down-regulated DE genes in the TB model group were related to cellular structural proteins and cellular functional proteins (Table 3), in which the structure and function proteins of alveolar epithelial cells account for a large proportion, for example, Sftpc, Sftpb, Sftpd, Sftpa1, Wfdc2, Sec14l3, Postn, Cldn5, Aqp5, Emp2, and Foxf1, etc.