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篇名
辨認New Delhi metallo-beta-lactamase 1 (NDM-1)的單株抗體之製備及分析
並列篇名
Generation and Characterization of Monoclonal Anti-NDM-1 Antibodies
作者 許瑋真李吉昕廖曉慧楊沂淵
中文摘要
本實驗,我們表現與純化Glutathione S-transferase (GST)與New Delhi metallo-beta-lactamase 1(NDM-1)組合而成的融合蛋白(GST-NDM-1)來免疫雞隻而產生多株IgY抗體。ELISA與Westernblot結果顯示IgY抗體可專一性辨認GST-NDM-1蛋白,也可以辨認切除GST蛋白後的NDM-1重組蛋白。利用phage display antibody technology建立兩個scFv抗體片段的抗體基因庫,經過篩選後,ELISA和Western blot結果顯示1個含有short linker (S7)和3個含有long linker (L6、L8、L13)的scFv可單獨辨認GST-NDM-1而不會辨認Glutathione S-transferase (GST)與人類enolase-1 (hENO-1)組合而成的融合蛋白(GST-hENO-1)。這結果顯示篩選出來的四個scFv抗體具有很好與NDM-1蛋白專一性結合的能力。我們相信這些具特異性的anti-NDM-1多株和單株抗體,對於帶有NDM-1基因的細菌感染的偵測以及檢驗試劑與藥物的開發上會有實質的幫助。
英文摘要
At present, only a handful drugs to inhibit the infection of bacteria with New Delhi metallo- beta-lactamase 1 (NDM-1) gene are available in clinical application, especially those against gram negative Enterobacteriaceae such as E. coli and K. pneumonia. Thus, developing the rapid diagnostic agents and new drugs to offer more efficient therapeutic strategies are urgently demanded. In this study, we chemically synthesized the full-length NDM-1 gene (810 bps) and cloned into expression vector. After expression and purification, recombinant GST-NDM-1 protein (~56 kDa) was used to immunize chicken intramuscularly to produce polyclonal IgY antibodies. Purified IgY antibodies specifically recognized the GST-NDM-1 fusion protein immobilized on ELISA wells. In addition to GST-NDM-1, they also showed significant binding activities to individual GST and NDM-1 protein on Western blots. In order to generate specific anti-NDM-1 monoclonal antibodies, we used phage display technology to construct two scFv libraries with short or long linker, which contained 1.2 × 10^7 and 8.4 × 10^6 transformants, respectively. After 4 rounds of panning procedure, we selected 28 clones to express scFv monoclonal antibodies and analyzed their binding activity against GST-NDM-1 or GST-hENO 1 protein. Among these 28 selected clones, 4 clones including one with short linker (S7) and three with long linker (L6, L8, L13) recognized GST-NDM-1 protein alone but not GST-hENO-1 on ELISA and Western blot, suggesting these 4 scFv antibodies possess specific binding activity against NDM-1 protein. We believed that these specific polyclonal IgY antibodies and scFv monoclonal antibodies could be applied in the development of diagnostic agents to prevent and cure the deadly infection of bacteria with NDM-1 gene in the future.
起訖頁 25-33
關鍵詞 IgY抗體scFv抗體New Delhi metallo-beta-lactamase 1IgY antibodiesscFv antibodies
刊名 生物醫學暨檢驗科學雜誌  
期數 201603 (28:1期)
出版單位 台灣醫事檢驗學會
該期刊-上一篇 使用Next Generation Sequence及SnapShot assay確認ABCG2及SLC22A12與國人痛風的相關性
該期刊-下一篇 運用OSCE教案評量及訓練醫檢師溝通技巧的能力
 

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