Zebrafish as Bioreactors to Produce Recombinant Protein

• Main Authors: Ping-Hsi Yang, 楊秉熹
• Other Authors: 蔡懷楨老師
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/09292473772887457165

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 96 === Zebrafish is an excellent alternative animal for using as a bioreactor because of short generation time, light-induced spawning, high fecundity, easy manipulation of gene transfer, and cheap culture system. Bovine lactoferricin possesses antimicrobial activity against a wide range of microorganisms, antiviral, antitumor and immunomodulatory activities. In this study, we generated transgenic zebrafish as a bioreactor to produce recombinant lactoferricin through whole body and eggs. An expression plasmid, in which lactoferricin-GFP was driven by zebrafish β-actin promoter, was microinjected into 2000 one-celled embryos. We selected 500 GFP-positive eggs as G0 transgenic founders, and crossed with wild- type individually. In total, 6 G0 lines which produced GFP-positive F1 offspring were generated. A 776-bp PCR-product was amplified, corresponding the amplification of lactoferricin-GFP transgene, from the genomic DNA extracted from 10 F1 transgenic fish. Furthermore, a recombinant lactoferricin-GFP protein with molecular masses of 29.2 KDa was positive hybridization with GFP antiserum, when the total proteins extracted from 50 F1 transgenic fish were subjected to western blot analysis. We chose one of the 6 F1 transgenic lines, ZBL-5, to cross with wild-type zebrafish and found that GFP was ubiquitously expressed in whole embryo of F2, starting from 1-cell stage. The GFP expression rate of the F2 transgenic embryos examined was 50.9 ± 2% (78/162, 64/121, 360/695), indicating that ZBLFB-5 is a stable heterozygotic transgenic line. In addition, in agar-well diffusion assay, we found thatthe bactericidal functional domain was enabled to release from lactoferricin-GFP fusion protein produced by transgenic zebrafish after it was digested with additional pepsin, and showed bactericidal efficacy. The bactericidal efficacy against Escherichia coli and Edwardsiella tarda from one heterozygotic embryo were equivalent to 0.1 μg and 0.03μg ampicillin respectively; against Aeromonas hydrophila was equivalent to 0.5 μg tetracycline. After we fed the transgenic embryos to zebrafish, fish were infected by immersion in water containing E. tarda. The survival rate after 7 days infection of zebrafish fed with 50 transgenic embryos was greatly higher than that fed with wild-type embryos, 87.5% (n=8) versus 0% (n=8), suggesting that feeding the lactoferricin-GFP containing transgenic embryos enables to protect fish against E. tarda infection. In conclusion, we generated stable transgenic zebrafish lines that enable to produce the functional recombinant lactoferricin in this study. This strategy may be highly potential application for aquaculture, pasturage and therapeutic treatment.