Zebrafish as a bioreactor to produce recombinant pathogen-resistant proteins
Date Issued
2009
Date
2009
Author(s)
Kao, Chia-Ling
Abstract
Abstract In this study, we used zebrafish as a bioreactor to produce the functional proteins that enabled fish to resist the infection of pathogens. There were two proteins we developed: one was lactoferricin (LFB), an antimicrobial peptide; another was the envelope protein VP28 of white spot syndrome virus (WSSV), a WSSV-resistant protein. A LFB-harboring transgenic line generated by Yang (2008), ZBL-5, expressed LFB-GFP driven by β-actin promoter. We proved that the extracts isolated from the GFP-positive embryos derived from F2 generation of ZBL-5 displayed the inhibition ability against Edwardsilla tarda. After pepsin digestion, a 72-hpf embryo from ZBL-5 produced about 0.355 ng of exogenous LFB-GFP, and its bactericidal efficacy was equivalent to that of 2.88 ng ampicillin. When we fed adult zebrafish with 50 embryos of ZBL-5 and then immersed in water containing 1×105 CFU/ml E. tarda for 7 days, the survival rate was as high as 75±12.5%. However, the survival rate of zebrafish which fed with 50 wild-type embryos and challenged with E. tarda at same condition was only 4 ± 7.2％. These results clearly proved that the LFB-GFP produced by transgenic line ZBL-5 exhibited an antimicrobial LFB domain to kill a freshwater pathogen. Meanwhile, we also generated zebrafish transgenic lines which produced VP28 to serve as oral vaccine against WSSV for P. monodon. Firstly, we constructed two expression plasmids, in which VP28-GFP was driven by either β-actin promoter (pBVPG) or heat-inducible zebrafish heat shock promoter 70/4 (pHVPG). After the NotI-cut pBVPG was microinjected into 1500 one-celled zebrafish embryos, selected 100 GFP-positive G0 transgenic founders, and crossed with wild-type individually. Four G0 lines which produced GFP-positive F1 offspring were generated. Similarly, the NotI-cut pHVPG was microinjected into 2000 one-celled embryos, selected 300 GFP-positive G0 transgenic founders, and crossed with wild-type individually. There were seven G0 lines which produced GFP-positive F1 offspring were generated. According to the appearance of GFP intensity, we selected a transgenic line, HVPG-16, for further study due to its GFP was highly expressed after heat shock induction. We extracted genomic DNA of F2 embryos from HVPG-16 and detected by PCR. A PCR-produce with molecular mass of 1417-bp was amplified, which was corresponding that of PCR product amplified from transgene fragment VP28-GFP. Furthermore, when the total proteins extracted from F2 2-month-old transgenic fish were subjected to western blot analysis using antiserum against GFP, two positive bands with 60 kDa and 70 kDa were found on the gel. The 60-kDa signal was corresponding to that of recombinant VP28-GFP protein. Interestingly, the GFP expression rate of the F2 transgenic embryos examined was 47% ( 211/225, 249/291), indicating that the single insertion of transgene in this stable heterozygotic transgenic line HVPG-16. Hence, in this study, we generated a transgenic zebrafish that produces the virus envelop protein VP28. The further study on the resistance of WSSV infection of tiger shrimp after feeding the feed powder mixed with VP28-containing embryos is in progress.
Subjects
zebrafish
WSSV
lactoferricin
SDGs
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