Journal: International journal of molecular sciences

Article Title: Generation and Characterization of Recombinant Pseudorabies Virus Delivering African Swine Fever Virus CD2v and p54 .

doi: 10.3390/ijms25010335

Figure Lengend Snippet: Figure 5. Production of anti-p54 and anti-CD2v-antibodies in mice. (a) Mice were infected with PRV-Fa, PRV-∆TK/∆gE, and PRV-∆TK-(CD2v)-∆gE-(p54) by IM injection into the leg (100 µL, 5 × 105 TCID50); PBS was used as a control. Post-infection, mice were sacrificed on day 3, and spleens were fixed in 4% paraformaldehyde. Then, paraffin-embedded sections were prepared and subjected to immunohistochemical staining for Flag-tag (top) and His-tag (bottom), representing the CD2v and p54 proteins, respectively. Scale bar, 100 µm. Insets (scale bar, 20 µm) represent an enlarged local area. The red arrow indicates cells presenting and expressing CD2v or p54 protein. (b,c) The p54-6×His-tag protein (~45 kD) was purified using a Ni-NTA beads column. Coomassie blue-stained SDS–PAGE (b) and Western blot (c) of samples from a typical purification of P54 protein from overex- pressed 293T cells. Lanes are labeled as follows: M, molecular-weight standards; 293T, untransfected 293T cells; T, total protein; FT, flowthrough (nonbound) from the Ni-NTA beads column; W, protein from the Ni-NTA beads column that was eluted in the wash fractions; P, purified protein after elution from the Ni-NTA beads column; B, Ni-NTA beads after protein elution. The red arrow indicates the target protein. (d) PRV-∆TK/∆gE and PRV-∆TK-(CD2v)-∆gE-(p54) were immunized into C57BL/6 mice via IM injection into the leg (100 µL, 5 × 105 TCID50). Serum samples were collected on day 7/14 post-immunization for p54- and CD2v-specific IgG detection via ELISA. The results demonstrate that mice immunized with PRV-R produced both p54- and CD2v-specific IgG. The data shown were obtained from five mice in each group. Error bars represent ± SD. Statistical analysis was performed with the 2-tailed unpaired Student t-test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: The purity of the recombinant protein was confirmed through Coomassie blue-stained SDS-PAGE and validated using Western blot analysis (probed with mouse anti-His-tag antibody, Proteintech, Wuhan, China, Cat No.: 66005-1-Ig).

Techniques: Infection, Injection, Control, Immunohistochemical staining, Staining, FLAG-tag, Expressing, Purification, SDS Page, Western Blot, Labeling, Molecular Weight, Enzyme-linked Immunosorbent Assay, Produced