利用斑馬魚作為生物反應器表現抗病蛋白

Zebrafish as a bioreactor to produce recombinant pathogen-resistant proteins

高佳玲(Chia-Ling Kao)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10694

摘要

摘要
本研究欲以斑馬魚作為生物反應器大量表現外源抗病蛋白，分別建立斑馬魚表現抗菌蛋白lactoferricin (LFB)及抗白點病毒 (white spot syndrome virus, WSSV)的膜鞘蛋白VP28兩種系統。先前楊 (2008)建立以β-actin promoter驅動LFB-GFP持續表現之轉殖品系ZBL-5。本實驗取有綠螢光表現的ZBL-5之F2胚胎經synthetic gastric acid作用使LFB釋出，結果發現具有殺E. tarda的效果，並且一顆來自ZBL-5品系72 hpf之胚胎所釋出的LFB相當於2.88 ng ampicillin的效力。而利用ELISA進行定量，可知平均一顆72 hpf的F2胚胎約含有0.355 ng的LFB-GFP。此外，餵50顆3-dpf ZBL-5的F2胚胎給斑馬魚成魚，之後浸泡在1 × 105 CFU/ml的E. tarda菌液中，七天後平均存活率約75± 12.5%，而相同條件下餵食wild type胚胎的斑馬魚其存活率僅約4 ± 7.2％。因此證明ZBL-5轉殖品系所表現的LFB-GFP經餵食後在魚的消化道中可釋出具有抵抗病原菌E. tarda感染功效的LFB。此外，本實驗也建立能表現WSSV膜鞘蛋白VP28的斑馬魚品系，以使應用於草蝦口服疫苗。首先構築兩種基因轉殖質體：分別利用β-actin promoter驅動VP28-GFP表現之pBVPG質體及經熱誘導後以zebrafish heat shock promoter 70/4驅動VP28-GFP之pHVPG質體。注射pBVPG質體到約1500顆的斑馬魚胚胎中，挑選其中100顆有綠螢光均勻分布表現的胚胎，經遺傳配對後獲得4個品系；注射pHVPG質體到約3000顆斑馬魚胚胎中，挑選經熱休克後其中300顆有綠螢光均勻表現的胚胎，經遺傳配對後獲得7個品系。根據綠螢光的表現強度，選用HVPG-16之品系進行後續實驗。HVPG-16 之F2子代胚胎經過熱誘導後約有47 % ( 211/225, 249/291)有綠螢光表現，抽取這些胚胎之genomic DNA，利用PCR方法，可偵測到約1.4 kb的 VP28-GFP之外源基因片段；並利用Western blot方法，以 GFP抗體進行偵測2個月大成魚的全蛋白，發現有60 kDa及70 kDa訊號出現，而60 kDa的訊號大小與VP28-GFP蛋白片段分子量相符合。因此，本研究證實斑馬魚可以穩定表現外源病毒膜鞘蛋白。未來進行使蝦子口服抗病毒蛋白之實驗以預期應用於水產養殖以減少疾病所造成的損失。

關鍵字

斑馬魚 ； 白點病毒 ； 牛乳鐵蛋白

並列摘要

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.

並列關鍵字

zebrafish ； WSSV ； lactoferricin

參考文獻

楊秉熹. (2008). Zebrafish as bioreactors to produce recombinant protein. 國立台灣大學分子與細胞生物學研究所碩士論文

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