Fig. 1.

CCL21 expression by S. typhimurium. Attenuated lipid A-negative S. typhimurium (pur⁻/msb⁻) were generated as previously described [6]. A cDNA encoding murine CCL21 (InvivoGen, San Diego, CA, USA) was inserted into pGEN206. This vector is a slightly modified form of pGEN222 [25] in which the parA locus has been exchanged with a parM and a parR locus. In addition, a repA locus has been introduced between the origin of replication and the multiple cloning sites. Expression of the 12 kD CCL21 protein was demonstrated by immunoblot analysis of cell lysates (“pellet”) or culture supernatants (“SUP”) from 10⁹ CFU of S. typhimurium containing either empty vector (Sal) or CCL21-bearing plasmid-transformed S. typhimurium (Sal + CCL21). Samples were normalized for total protein content, resuspended in Laemmli sample buffer and subjected to SDS-PAGE followed by immunoblotting with a polyclonal goat anti-CCL21 antibody (Abcam, Cambridge, MA, USA). Results are representative of three independent experiments