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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >SEQUENCE ANALYSIS OF WHEAT GRAIN ALLERGENS SEPARATED BY TWO-DIMENSIONAL ELECTROPHORESIS WITH IMMOBILIZED PH GRADIENTS
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SEQUENCE ANALYSIS OF WHEAT GRAIN ALLERGENS SEPARATED BY TWO-DIMENSIONAL ELECTROPHORESIS WITH IMMOBILIZED PH GRADIENTS

机译：固定化pH梯度二维电泳分离小麦粒变应原的序列分析

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Micropreparative two-dimensional (2-D) gel electrophoresis with immobilized pH gradients (4-8) in the first dimension (IPG-DALT) was optimized for the separation of salt-soluble wheat grain proteins associated with bakers' asthma disease. The resolved polypeptides were electroblotted onto a polyvinylidene difluoride (PVDF) membrane and incubated with the pooled sera from four asthmatic bakers. Bound IgE was demonstrated by alkaline phosphatase conjugated anti-human IgE. Major IgE binding was detected in the 27 kDa, 37 kDa and, to a lesser extent, in the 14-18 kDa area of the 2-D immunoblots, respectively. Since the main purpose of our study was to determine the N-terminal amino acid sequences of the major wheat grain allergens, N-terminal sequencing was performed for six out of a total of eleven major allergens located in the 27 kDa area, for one out of two 37 kDa allergens, and for two out of four 14-18 kDa allergens. Our results revealed that two of the 27 kDa polypeptides are clearly related to several Acyl-CoA oxidase variants of barley and rice, whereas no significant homologies were found for the remaining four 27 kDa allergens analyzed. The N-terminus of the 37 kDa allergen appeared to be blocked so that no sequence information was obtained, while the two 14-18 kDa allergens analyzed were identified as members of the wheat a-amylase-inhibitor family. [References: 21]
机译：微制备二维（2-D）凝胶电泳具有固定的pH梯度（4-8），且在第一维（IPG-DALT）被优化用于分离与面包师哮喘病相关的盐溶性小麦籽粒蛋白。将分离的多肽电印迹到聚偏二氟乙烯（PVDF）膜上，并与来自四个哮喘面包师的合并血清一起孵育。结合碱性磷酸酶的抗人IgE证明了结合的IgE。在二维免疫印迹的27 kDa，37 kDa和较小程度的14-18 kDa区域分别检测到主要IgE结合。由于我们研究的主要目的是确定主要小麦籽粒过敏原的N末端氨基酸序列，因此对位于27 kDa地区的11种主要过敏原中的6种进行了N末端测序， 2种37 kDa的过敏原，4种14-18 kDa的过敏原中的2种。我们的结果表明，27 kDa的多肽中有两个明显与大麦和水稻的几种酰基辅酶A氧化酶变体相关，而其余4种27 kDa的过敏原均未发现明显的同源性。 37 kDa过敏原的N端似乎被阻断，因此未获得序列信息，而分析的两个14-18 kDa过敏原被鉴定为小麦α-淀粉酶抑制剂家族的成员。 [参考：21]

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