Silencing the major apple allergen Mal d 1 by using the RNA interference approach
Silencing the major apple allergen Mal d 1 by using the RNA interference approach
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DOI：
10.1016/j.jaci.2004.10.014
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发表时间：
2005-02-01
影响因子：
14.2
通讯作者：
van Ree, R
中科院分区：
文献类型：
--
作者：
Gilissen, LJWJ;Bolhaar, STH;van Ree, R
背景：在桦树花粉流行的地区，苹果过敏主要由抗Mal d 1的IgE抗体引起。显著降低Mal d 1水平的苹果将使这些地区的大多数患者食用苹果而不产生过敏反应。目的：本研究的目的是通过RNA干扰技术抑制苹果植株中Mal d 1的表达。方法：在体外生长的苹果小植株用编码内含子剪接的发夹RNA的构建体转化，所述内含子剪接的发夹RNA含有Mal d 1-由Mal d 1特异性内含子序列分隔的特异性反向重复序列。通过PCR检查转化体中构建体的存在。通过对3名苹果过敏患者进行皮肤点刺试验和用Mal d I反应性mAb和抗Mal d 1的IgE抗体进行免疫印迹来监测Mal d 1在叶片中的表达。通过PCR，在9个选择的植株中的6个中，证明了基因沉默构建体的存在。通过皮肤点刺试验，表明野生型小植株具有比5个转化体显著（P <0.05）更高的变应原性。通过免疫印迹证实Mal d 1表达的减少。在野生型和不成功的转化体中，用Mal d 1反应性mAb 5 H8在预期的表观M-r 17 kDa处检测到强条带。该条带在携带基因沉默构建体的转化体中几乎不存在。与人IgE抗体，同样的observations made.Conclusions：Mal d 1的表达成功地减少了RNA干扰。这转化为显著降低的体内变应原性。这些观察结果支持了通过基因沉默生产对Mal d 1低变应原性的苹果的可行性。
Background: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions.Objective: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference.Methods: In vitro-grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d I-reactive mAb and with IgE antibodies against Mal d 1.Results: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly (P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M-r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made.Conclusions: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.