利用動物性浮游生物作為生物反應器表現外源抗菌蛋白

Transgenic zooplankton as a bioreactor to produce recombinant anti-microbial proteins

陳弘昕(Hung-Sin Chen)

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(2014年)

https://doi.org/10.6342/NTU.2014.10112

摘要

中文摘要
餌料生物豐年蝦可作為外源蛋白載體，提供養殖魚類額外生長激素或抗菌胜肽。然而，目前所用豐年蝦休電穿孔轉殖條件所能達到孵化率及轉染效率(Transfection efficiency)較低，加上綠螢光蛋白(GFP)作為報導基因容易受豐年蝦內生性蝦紅素所產生紅螢光干擾，造成綠螢光訊號不易觀察，增加穩定遺傳品系建立之困難度及成本。因此，本實驗藉由調整電場強度及脈衝時間的方式，建立較不影響休眠卵孵化率之豐年蝦基因轉殖系統；同時，本實驗採用藍螢光蛋白(N158S)基因當報導基因，因為藍螢光蛋白有特殊的波長以及較高之量子產率(Quantum yield)性質。除轉殖系統改良外，本實驗也將藍螢光基因作為selective plasmid，與另一帶有海鞘抗菌胜肽(Ci-MAM-A)cDNA及天蠶素(Cecropin)cDNA之融合型抗菌胜肽質體pCMV-MB-Ci-Ce 作為target plasmid，同時轉殖入豐年蝦體內表現，藉藍螢光訊號觀察建立穩定遺傳雙基因之轉殖品系。利用改良轉殖條件進行休眠卵轉殖後，在八次電穿孔共同轉殖實驗中共得529隻體軸表現藍螢光之無節幼蟲，其表現率為21.45%(529/2466)，進而取50隻體軸表現強藍螢光個體(F0)與野生型豐年蝦進行互配得到五組體軸表現強藍螢光訊號之F1子代。將五組F1世代與野生型豐年蝦進行互配後，得到四組體軸仍穩定表現藍螢光之F2子代，並針對F1及F2世代進行PCR檢測，篩出兩株穩定含有藍螢光基因與抗菌胜肽基因之遺傳品系CCNS 9及CCNS 10。選擇生長及繁殖情況較快速之CCNS 10的F2子代與野生型互配而得F3世代。利用western blotting 偵測轉殖品系蛋白質萃取液後，可在17 kD處偵測到Ci-MAM-A-Cecropin蛋白質條帶，表示轉殖品系的確表現外源重組抗菌蛋白。另外，透過外加胃蛋白酶(Pepsin)處理及抑菌環試驗，結果顯示CCNS 10遺傳品系之F3世代單隻成體豐年蝦所含的外源抗菌胜肽，其抑制大腸桿菌能力約等同於0.00233 μg之Ampicillin。因此，我們成功地建立可以穩定表達外來藍螢光基因與抗菌胜肽基因之豐年蝦轉殖品系，並以證實其具有抑制大腸桿菌之能力，未來可進一步檢測此品系對其他微生物之抑制效力，並可應用在水產養殖上以口服方式幫助養殖魚類抵抗外界病菌。

關鍵字

豐年蝦 ； 抗菌蛋白 ； 生物反應器

並列摘要

Abstract
Brine shrimp (Artemia sp.) is a potential suitable bioreactor for applying cultured finfish growth hormones or antimicrobial peptides to cultured finfish. However, low transformation efficiency and hard-to-distinguished GFP signal resulted in the establishment of Artemia transgenesis being difficult and time consuming. In this study, we developed a new electroporation condition by modifying the field strength and plus length to achieve a higher transformation efficiency. We also used a unique selective marker which has different absorption spectrum and higher quantum yield than those of green fluorescence protein for screening the transgenic nauplius of Artemia. After the Artemia cysts were decapsulated, they were then electroporated with two plasmids, a selective plasmid containing selective protein and a target plasmid containing antimicrobial peptides. The transient expression rate of selective plasmid was 21.45% (529 out of 2466 examined). We selected 50 F0 individuals to mate with the wide-type strain and generated F1 offspring. Among F1 generation, we selected 5 strains with highly expressed signal of selective protein to generate F2 offspring. Genomic DNA extracted from the F1 and F2 were checked by PCR detection and found that they were all positive. Furthermore, two stable transgenic lines of Artemia sp. were generated, named CCNS 9 and CCNS 10. We confirmed that the recombinant protein with molecular weight of 17 kD was produced by F3 offspring derived from transgenic Artemia CCNS 10 by Western blotting analysis. In addition, the agar diffusion test demonstrated that the antimicrobial activity of the recombinant proteins produced by CCNS 10 was equivalent to the potency generated by 0.002 μg of Ampicillin. In conclusion, this study showed that we successfully generated a stable line of transgenic Artemia which possessing antimicrobial activity to E. coli. This transgenic line may have the potential in aquaculture to provide the cultured finfish antimicrobial peptide by oral treatment.
Using the new electroporation system and selective marker, we generated a transgenic Artemia line expressing recombinant antimicrobial peptides.

並列關鍵字

Artemia ； antimicrobial peptides ； bioreactor

參考文獻

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