基因轉殖斑馬魚作為生物反應器表現外源重組蛋白質

Abstract

斑馬魚 (zebrafish) 成為新興的生物反應器，因為生命周期較短，終年可產卵，產卵數多，基因轉殖容易，養殖設備簡單且成本便宜。Bovine lactoferricin具有廣泛的抗微生物活性，包括細菌與真菌。另外也具有抗病毒、抗癌細胞的活性，還有調節免疫的能力。於是欲使用斑馬魚作為生物反應器，於斑馬魚體及卵中來製造重組 lactoferricin 蛋白質，將來能提供養殖、畜產與醫療的用途。本研究先構築由β-actin promoter來驅動lactoferricin-GFP基因的片段，然後透過顯微注射將此片段轉殖到2000個左右斑馬魚胚胎中，並利用綠螢光篩選出500隻可能之G0親代。育成後與野生型配對，在其中篩選出6隻能產下綠螢光F1的G0，接著使用PCR檢測其中10隻轉殖魚F1胚胎的genomic DNA，可夾取出776 bp轉殖進來的lactoferricin-GFP片段。使用Western blot偵測50隻轉殖魚F1胚胎的total proteins，可偵測到29.2 KD 的lactoferricin-GFP重組蛋白質。在6個轉殖品系中，挑選ZBL-5品系的轉殖魚F1與野生型配對，產下的 F2子代胚胎在1- cell時已可觀察到有綠螢光的表現，隨時間增加全身綠螢光的表現有持續累積的現象，並且胚胎中表現綠螢光的比例為50.9% ± 2% (78/162, 64/121, 360/695)，顯示為穩定的heterozygotic 轉殖魚品系。此轉殖魚產生之lactoferricin-GFP蛋白質，可經外加pepsin 切下產生具有抗菌活性的 functional domain，經實驗結果得知ㄧ隻72 hpf 表現綠螢光的F2轉殖魚胚胎所含有的lactoferricin，在agar plate顯示出其殺大腸桿菌 (Escherichia coli)、遲鈍愛德華氏菌(Edwardsiella tarda) 與親水性產氣單胞菌(Aeromonas hydrophila) 的效力分別相當於0.1μg ampicillin、0.03μg ampicillin 與0.5 μg tetracycline的劑量。並且將50隻帶有lactoferricin-GFP的胚胎 (72hpf) 餵食斑馬魚，八小時後浸泡於E. tarda 菌液中進行感染實驗。發現七天後餵食相同數量 wild-type胚胎的斑馬魚存活率由0% (n=8) 提升為87.5%(n=8)。顯示餵食帶有lactoferricin-GFP的胚胎，確實可在腸胃道發揮其生物活性幫助斑馬魚抵抗E. tarda的感染、降低魚隻致死率。總結，本研究得到可以產製具有功能的lactoferricin外來蛋白質之穩定遺傳品系，將來可應用在水產養殖、畜牧與醫療等用途。

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.