Fig. 2
Construction of nisin-inducible MuIFNλ3 expression cassette and protein expression by rL. lactis. Recombinant plasmid expression cassette (pSec-MuIFNλ3) encoding signal peptide (sp) of lactococcal USP45 gene sequence (75 bp) followed by MuIFNλ3 gene (582 bp) flanked with NsiI and NheI restriction sites (a). The graphical representation of the nisin-inducible expression and secretion of rMuIFNλ3 protein from rL. lactis cells (b). Agarose gel image showing specific amplification of rMuIFNλ3 gene with a size of 582 bp following colony PCR of electro-transformed L. lactis cells (NZ9000) (M: Marker; 1,2: rL. lactis colonies) (c). The comparative growth profile of WT L. lactis, nisin-induced or uninduced rL. lactis cells. Altered (reduced) growth profile of nisin-induced rL. lactis (dotted line) cells compared to uninduced rL. lactis or empty L. lactis (solid lines). “Arrow” indicates the time of nisin induction (d). Western blot analysis of TCA precipitated culture supernatant of nisin-induced rL. lactis cells probed with rabbit anti-rMuIFNλ3 antisera detect ~ 22 kDa protein corresponding to the size of MuIFNλ3 (e). Quantification of MuIFNλ3 protein secreted in the culture supernatants of rL. lactis by indirect ELISA suggests ~ 2.5 ng/mL of protein secreted by 1 × 109 rL. lactis cells at 3 h post-induction. Dot “♦·” indicates the approximate quantification of protein secreted by induced rL. lactis (f)