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Title: | 基因轉殖豐年蝦表現外源重組蛋白 Transgenic Artemia as a Bioreactor to Produce Recombinant Proteins |
Authors: | Ting-Lieh Hsieh 謝庭列 |
Advisor: | 蔡懷楨(Huai-Jen Tsai) |
Keyword: | 豐年蝦,抗菌蛋白,生長激素, Artemia,Antimicrobial peptide,Growth hormone, |
Publication Year : | 2012 |
Degree: | 碩士 |
Abstract: | 本研究欲採用甲殼類豐年蝦作為生物反應器。於豐年蝦體中製造魚類的生長激素 (ypGH)及牛的乳鐵蛋白,以提供魚類抗病及促進成長用途。首先,先構築可以表現外來基因的表現質體。透過電穿孔方式轉殖至豐年蝦中,利用綠螢光篩選出100隻G0親代,並與野生種配對,之後利用PCR及螢光篩選出帶有綠螢光的YL6及YL8兩個品系 (strain)的F1子代,育成F2子代,並將F2個體進行自交後,產出F3子代。利用PCR檢測這些F3個體,可偵測到1.3 k的外來基因片段。抽取YL6及YL8兩品系F3子代蛋白質,以tGFP抗體都可偵測到50 kDa的外來重組蛋白質。若以ELISA偵測無節幼蟲帶有的外來重組蛋白質含量,每隻YL6的為0.0011 μg及每隻YL8的為0.0004 μg。另外使用pepsin進行對YL6轉殖豐年蝦蛋白質進行處理,以tGFP抗體可偵測到被pepsin截切後的27 kDa tGFP蛋白質,而以ypGH抗體可以偵測到21 kDa ypGH蛋白質,表示外來重組蛋白確實存在。在透過外加pepsin切下會產生抗菌活性,經抑菌環實驗結果得知YL6每一隻F3轉殖豐年蝦在agar plate顯示出其殺大腸桿菌 (Escherichia coli)的效力。總結,本研究得到可以產製外來蛋白質之穩定遺傳品系豐年蝦,將來可進一步於in vivo進行生物活性試驗,並可應用於水產養殖及醫療等用途。 In this study, we use Artemia sp. as a bioreactor to produce recombinant proteins through whole body. We constructed an expression plasmid and introduced into Artemia by electroporation to generate the transgenic Artemia. We selected 100 GFP-positive Artemia as G0 transgenic founders, and crossed with wild-type individually. In total, two G0 lines which produced GFP-positive F1 offspring were generated. Among F1 offspring, we crossed GFP- positive F1 with wild-type individually to generate F2, and thereof F3 generations of Artemia. Genomic DNAs were extracted from the G0, F1, F2 and F3 individuals and checked with PCR detection, a 1.3 k PCR-product was amplified. Two stable transgenic Artemia lines were screened, named YL6 and YL8. Moreover, a recombinant protein with molecular masses of 50 kDa from transgenic Artemia was positive hybridization with tGFP antiserum. The concentration of recombinant proteins produced by one homozygotic nauplii of YL6 and YL8 were 0.0011 μg and 0.0004 μg, respectively, by ELISA. In addition, in agar-well diffusion assay, the bactericidal functional domain was able to release from fusion protein after it was digested with pepsin, suggesting that the bactericidal efficacy against Escherichia coli from one transgenic Artemia YL6 was positive. In conclusion, this study showed that we successfully generated stable transgenic Artemia lines which may be potentially applied for aquaculture and therapeutic treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16443 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 分子與細胞生物學研究所 |
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