Figure 4.

Display of a CD40-targeting peptide on the phage capsid results in an enhancement of phage-mediated DNA uptake transfer into RAW 264.7 cells. (A) RAW 264.7 cells were incubated with phage particles at an MOI of 1 × 10⁶ p.f.u. After an initial centrifugal enhancement step (1200 g, 1 h), cells were incubated with the phage for 48 h at 37°C, and then washed and lysed. Analysis of lambda genome copy number was conducted using 10 ng of total input cellular DNA prepared from the cell lysates. DNA copy number was slightly, but significantly (P < 0.05), greater in cells which were transduced with phage particles that displayed gpV-CD40 versus unmodified phage (gpD). The bi-functional phage, combining gpV-CD40 with gpD-UBHA, further increased lambda genome copy number (P < 0.001, when compared to phage displaying gpD-UBHA or gpV-CD40 alone). Surface display of the UBHA peptide did not lead to a statistically significant increase in the number of phage genome copies per cell, when compared to unmodified phage particles (gpD). Statistical analysis was performed using one-way ANOVA with Tukey's post-test; significance was taken as P < 0.05. (B) RAW 264.7 cells were stained with an anti-CD40-PE antibody or an isotype control antibody, and stained cells were analyzed by flow cytometry. The darkly shaded curve represents staining with the isotype control antibody, and the unshaded curve represents staining of cells with the CD40-specific antibody.