Transgenic zebrafish eggs containing bactericidal peptide is a novel food supplement enhancing resistance to pathogenic infection of fish.
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.
Lin
Cheng-Yung
CY
Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan, ROC.
Yang
Ping-Hsi
PH
Kao
Chia-Ling
CL
Huang
Han-I
HI
Tsai
Huai-Jen
HJ
D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019149:Bioreactors; D015153:Blotting, Western; D002417:Cattle; D019587:Dietary Supplements; D004531:Eggs; D005393:Fish Diseases / Q000276:immunology* / Q000401:mortality / Q000517:prevention & control; D006090:Gram-Negative Bacteria / Q000187:drug effects; D016905:Gram-Negative Bacterial Infections / Q000276:immunology / Q000401:mortality / Q000517:prevention & control / Q000662:veterinary*; D007113:Immunity, Innate / Q000187:drug effects*; D007781:Lactoferrin / Q000235:genetics / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism / Q000382:microbiology
D000199:Actins; D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; C082171:lactoferricin B; D007781:Lactoferrin