lactoferricin-b and Gram-Negative-Bacterial-Infections

Zebrafish (Danio rerio) was used as a bioreactor to produce bovine lactoferricin (LFB), which has wide-ranging antimicrobial activity. We constructed an expression plasmid in which LFB was fused with green fluorescent protein (GFP) and driven by zebrafish beta-actin promoter. After microinjection, six transgenic founders were screened on the basis of GFP appearance. Among them, a stable ZBL-5 line was selected by the ubiquitous and strong expression of GFP. Using PCR and Western blot analysis, we confirmed that the recombinant LFB-GFP protein was produced by the F2 progeny derived from the ZBL-5 line. The bactericidal agar plate assay proved that the functional domain of LFB was released from the LFB-GFP fusion protein, resulting in strong bactericidal activity against Escherichia coli, Edwardsiella tarda and Aeromonas hydrophila. Furthermore, adult zebrafish were given one feeding of fifty 72-hpf transgenic embryos. The treated fish were then immersed in freshwater containing 1 x 10(5) CFU ml(-1)E. tarda for 7 days. The survival rate of the treated zebrafish was significantly higher than that of fish fed with fifty wild-type embryos (75 +/- 12.5% versus 4 +/- 7.2%). This line of evidence suggested that pathogen resistance can be enhanced by using transgenic embryos containing LFB-GFP as a food supplement for fish, while, at the same time, reducing the demand of chemical antibiotics.

Topics: Actins; Animals; Animals, Genetically Modified; Anti-Bacterial Agents; Bioreactors; Blotting, Western; Cattle; Dietary Supplements; Eggs; Fish Diseases; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Immunity, Innate; Lactoferrin; Polymerase Chain Reaction; Recombinant Proteins; Zebrafish