檢測植酸酶轉基因米與非轉基因米中主要過敏原蛋白質之含量   全文下載

Detection of Major Rice Allergenic Proteins in Phytase-Transgenic and Non-Transgenic Rice

米中14-16kDa過敏原蛋白已被研究證實屬於多基因家族之產物，並且是米過敏病人血清中IgE反應最常見之過敏蛋白。本研究之目的在分析米中主要過敏原蛋白質（14-16kDa rice allergenic protein, RAP）之含量在植酸?轉基因米與母本米（台農67號）間是否有差異。為了探討外來基因對母本稻米是否造成致敏性改變，在其上市前需做安全性檢測評估，以釐清人們之疑慮。首先，利用逆轉錄聚合?連鎖反應（reverse transcription-poly-merase chain reaction, RT-PCR）擴增米中16 kDa RAP基因-RA17之cDnA，將之構築成pET-29a(+)-RA17表現載體，並送入Escherichia coli BL21(DE3)蛋白質表現宿主中。宿主經誘導表現重組蛋白RA17，再以ni2+-nTA resin 管柱分離得到純化的重組蛋白，將此重組蛋白注射至大白兔體內後取得多株抗體。利用此抗體進行西方墨漬法及酵素免疫分析法分析米中主要過敏原蛋白質之含量。由西方墨漬法（Western blotting）測得的結果顯示，植酸?轉基因米所含的主要過敏原蛋白質之含量為母本米之96.3%；酵素免疫分析法（ELISA）分析結果，主要過敏原蛋白質含量在母本米（nR）為1.73 ± 0.09 mg/g 、植酸?轉基因米（GR）為1.66 ± 0.08 mg/g，經統計分析結果兩者間並無顯著差異（p > 0.05）。顯示植酸?轉基因米中主要過敏原蛋白質，並不會因外來植酸?基因而造成其過敏原蛋白質含量的增加。"

The 14-16 kDa rice allergenic proteins have been reported to be the products of a multigene family, and react most frequently with IgE in serum of rice allergic patients. The aim of this study was to assay the content of endogenous 14-16 kDa rice major allergenic proteins in both phytase-transgenic (GR) and non-transgenic rice (NR) (wild-type, Oryza sative L. cv. Tainung 67). We want to investigate whether the foreign gene will change the allergenicity of rice or not, should be evaluated in order to clarify its pre-market safety. First, the RA17 gene encoding 16 kDa rice allergenic protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), and cloned into the expression vector pET-29a(+). The constructed expression vector, pET-29a(+)-RA17, was transformed into Escherichia coli BL21 (DE3). The host strain was induced to express recombinant RA17, which was purified by Ni2+-NTA resin column and used as the antigen for the production of a polyclonal antibody via rabbits. We used the antibody as a detective tool of western blotting and enzyme-linked immunosorbent assay (ELISA) methods. The western blotting analysis showed that the content of major allergenic proteins in phytase-transgenic rice was 96.3% of that in wild-type rice. The content of 14-16 kDa allergens estimated by ELISA were 1.73 ± 0.09 (NR) and 1.66 ± 0.08 mg/g (GR). The results indicated that the level of 14-16 kDa allergens was not significantly different (p > 0.05) between wild-type rice and phytase-transgenic rice, and the transformation of phytase gene into rice plant did not enhance the content of rice major allergenic proteins in transgenic rice.