*3.5. Analysis of Target Gene Expression*

The genes expression of DNA vaccines was evaluated with two methods: specific mRNA synthesis assay and immunostaining of the transfected cells. To evaluate synthesis of specific mRNA, we isolated total RNA from 293T cells transfected with plasmids pEV.CTL and pEV.Th and obtained cDNA in RT. The obtained cDNA was used to carry out PCR using pairs of primers (fCTL, rCTL) and (fTh, rTh) to genes *EV.CTL* and *EV.Th*, respectively.

The results in Figure 2 demonstrate that the sizes of the amplified fragments correspond to the theoretically calculated sizes of amplification products, i.e., 891 bps for *EV.CTL* gene and 495 bps for *EV.Th* gene. Similar PCR fragments were obtained when using initial target plasmids pEV.CTL and pEV.Th (positive control) as a matrix. The findings indicate presence of specific mRNA in the total cell RNA fraction.

**Figure 2.** Electrophoregram on 1% agarose gel of PCR products: 1 - Molecular weight marker (M12, SibEnzyme); 2 and 3—PCR fragments of 831 and 495 bps obtained using cDNA as a matrix with primers (fCTL, rCTL) and (fTh, rTh), respectively; 4 and 8—The results of PCR with primers (fCTL, rCTL) and (fTh, rTh) and total RNA isolated from 293T cells transfected with plasmids pEV.CTL and pEV.Th, respectively (without reverse transcription; control for the absence of target plasmids in isolated samples of total RNA); 5—Molecular weight marker (M15, SibEnzyme); 6 and 7—PCR fragments obtained using plasmids pEV.CTL and pEV.Th as a matrix, respectively (positive control).

Immunohistochemical staining of cells transfected with pEV.CTL and pEV.Th plasmids was evaluated using MAT 29F2/30A6 antibodies to EPFRDYVDRFYKTL marker epitope, included in all constructs. The results depicted in Figure 3 demonstrate the presence of specific proteins. The findings confirm the expression of the target genes both at the level of transcription and translation.

**Figure 3.** Evidence of genes expression in cells transfected with plasmids pEV.CTL and pEV.Th by immunohistochemical staining. (**a**) 293T-cells transfected with p*EV.CTL* plasmid. (**b**) 293T-cells transfected with p*EV.Th* plasmid. (**c**) 293T-cells transfected with pcDNA3.1 vector plasmid.
