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標題: 芒果主要過敏原 Man i 1 基因定序與重組蛋白表現及建立酵素連結免疫法與側層流分析法以偵測食品中Man i 1芒果過敏原
Cloning and expression of major mango allergen- Man i 1, and development of enzyme-linked immunosorbent assay and lateral flow assay for detecting Man i 1 in foods
作者: 蔡汶哲
Wen-Che Tsai
關鍵字: 芒果過敏原;Man i 1;重組蛋白;酵素連結免疫法;側層流分析法;mango allergen;Man i 1;recombinant protein;an enzyme-linked immunosorbent assay;a lateral flow assay
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近幾年全球水果過敏人口逐漸增加,而芒果在台灣為主要的水果過敏原,故在食藥署規定的過敏原標示中芒果屬於「強制標示」,即食品若含有芒果成分必須在包裝上標示以提醒消費者。至今有關芒果主要過敏原Man i 1的研究資訊較少,僅知等電點與分子量。本研究先以3' RACE和5'RACE獲得Man i 1 cDNA 全部序列(1,024 bp),並將之構築在表現質體- pET-21a(+)以Escherichia coli BL21 (DE3)系統表現。實驗結果顯示最佳表現條件為在16oC及0.1 mM IPTG的條件下誘導20小時可以達到最大量的可溶性重組蛋白。再以Ni-NTA管柱純化,藉由anti-histidine 抗體與芒果過敏者的血清確認純化的蛋白質確實含有histidine與免疫反應,並以LC/MS/MS定出此重組蛋白的部分氨基酸序列,此結果涵蓋cDNA所推演出氨基酸序列的70%,由以上結果可確認本系統所表現出的重組蛋白即為rMan i 1,且可以被芒果過敏者的血清所辨識。此外,以此系統大量表現以及純化rMan i1,將之作為免疫原以製備單株與多株抗體,以及建立兩種快篩方法,以檢測食品中的芒果過敏原殘留。
為建立一個三明治酵素連結免疫法 (sandwich ELISA, sELISA)偵測食品中的Man i 1,1多株抗體、2株胜肽抗體與2株單株抗體進行配對,得到高訊號的抗體組合為以單株抗體(2G5 mAb) 作為捕獲抗體而多株抗體(anti-Man i 1 pAb)作為偵測抗體。優化sELISA條件後,評估sELISA的專一性、靈敏度、再現性和準確性。結果顯示sELISA在12種水果萃取物中,對芒果萃取物具有良好的專一性,另外其具有良好靈敏度,檢測極限為3.9 ng/mL。在批內與批間試驗中,其變異係數分別為5.5-11.7%和7.4-12.4%,故具有良好的再現性和準確性。最後以sELISA分析35種市面購買的食品樣品,其結果並與西方墨點法進行比對,結果顯示該開發的sELISA與西方墨點法具有良好的一致性,沒有假陽性或假陰性結果。共有6款食物產品含有芒果過敏原。這些實驗結果顯示sELISA可應用在檢測主要芒果過敏原-Man i 1。
另外建立側層流試紙系統檢測芒果過敏原Man i 1,其中探討金球製備條件、硝化纖維膜種類、測試線上抗體濃度、塗佈及阻抗溶液成分組成之條件對結果的影響。結果顯示免疫製備條件為10 mM 碳酸鉀與250 µg/mL的anti-rMan i 1 多株抗體混合,而側層流系統的最佳製備條件是在AE99硝化纖維膜上的測試線塗佈4 µg/cm anti-2G5單株抗體,並在結合墊加入10 µL免疫金球,而塗佈及阻抗溶液成分別為含有5%甲醇磷酸鹽緩衝溶液及磷酸鹽緩衝溶液(1% casein、5% sucrose、0.05% Tween 20)。評估側層流系統的靈敏度結果顯示檢測極限在PBS系統為0.097 µg/mL rMan i 1,而在芒果萃液的檢測極限為4.9 µg/mL芒果蛋白粗萃物,整體反應時間約20分鐘,此側層流檢測系統反應時間短、靈敏度高且專一性高,對於檢測芒果過敏原-Man i 1具潛在發展性,未來評估其再現性和準確性,及檢測食物樣品中芒果過敏原。

In recent years, the number of people around the world who suffer from fruit allergies has increased. In Taiwan, mango is a major allergic fruit and thus it is officially required that mango must be labeled on the package of the food may contain mango allergens. Mango is a tropical fruit that can induce anaphylaxis, and the major mango allergens have been identified as Man i 1. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3' and 5' RACE assays were used to identify the cDNA fragment of Man i 1 (1,024 bp), which was further analyzed by DNA sequencing. Subsequently, the full length of Man i 1 cDNA was amplified and inserted into a pET-21a(+) vector, and the rMan i 1 was expressed through Escherichia coli BL21 (DE3). The conditions for the expression of rMan i 1, including IPTG concentrations, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16oC for 20 hours. The His-tagged rMan i 1 was purified using Ni-NTA agarose from the supernatant of the cell lysate and its identity was verified using an anti-histidine antibody, the serum of a mango-allergic person, and LC/MS/MS. The rMan i 1 was also proofed that it had glyceraldehyde 3-phosphate dehydrogenase activity. Finally, an E. coli expression system of rMan i 1 was established, and rMan i 1 as antigen for the production of antibodies to develop sandwich ELISA (sELISA) and lateral flow assay (LFA) for the detection of mango allergen- Man i 1.
To developed a sELISA to detect the residues of Man i 1 in food samples, a variety of antibodies including polyclone antibodies, monoclonal antibodies, and anti-peptide antibodies against Man i 1 were generated and paired to obtain a high assay signal. After conditions of the sELISA method were optimized, the sensitivity, specificity, reproducibility, and accuracy of this developed sELISA were evaluated. The optimal sELISA where 2G5 mAb was as capture Ab and anti-Man i 1 pAb as detection Ab had good specificity to mango extract among 12 fruits extracts and had good sensitivity with detention limit of 3.9 ng/mL. Moreover, the coefficient of variants (%) of the intra- and inter-assay was 5.5~11.7% % and 7.4~12.4 %. The developed sELISA had great reproducibility and precision. Additionally, the performance of the developed assay was compared with that of Western blot when analyzing three-five commercial food samples. Six food products with mango allergen were detected by sELISA. These features demonstrate a promising application of the developed sELISA in detecting major mango allergen - Man i 1 in foods efficiently and reliably.
To develop a LFA to detect the residues of Man i 1 in food samples, types of nitrocellulose membrane, antibody density on the test line, and the compositions of coating buffer and blocking buffer were investigated. The optimized condition for preparing immunogold nanoparticles were by adjusting pH with 10 mM of K2CO3 and then conjugating with 250 µg/mL of anti-Man i 1 antibody. The optimal condition for preparation of LFA is by coating 4 µg/cm of 2G5 mAb prepared in PBS with 5% methanol on the test line of AE 99 nitrocellulose membrane. The composition of blocking buffer for the conjugate pad was 1% casein, 5% sucrose, 0.05% Tween 20 in PBS. The detection limit of this developed assay was 0.097 µg/mL of rMan i 1 in PBS and 4.9 µg/mL of crude mango protein extract with the reaction time about 20 minutes. This assay gave a good result in short time and had good sensitivity and spicifity as analyzing mango allergen- Man i 1. It has potential to be as rapid assay for detecting mango allergen in foods. In the future, the reproducibility, and accuracy of the developed LFA will be estimated, as well as its feasibility in detecting mango allergen residue in commercial food products will be testified.
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