Viquez O M, Summer C G, Dodo H W
Department of Food and Animal Sciences, Alabama A&M University, Normal, AL 35762, USA.
J Allergy Clin Immunol. 2001 Apr;107(4):713-7. doi: 10.1067/mai.2001.113522.
Peanuts have been identified as potent food allergens responsible for life-threatening IgE reactions among hypersensitive individuals. With the current increase of peanut allergies, there is an urgent need to molecularly characterize the genes encoding the target proteins and to understand the nature of their regulation.
The objectives of this study were to isolate, sequence, and characterize at least one full-length genomic clone encoding the major peanut allergen Ara h 2.
A peanut genomic library, constructed in a Lambda Fix II vector, was screened with an 80-bp oligonucleotide probe constructed on the basis of the 5' end of a published Ara h 2 cDNA partial sequence. One putative positive lambda clone was isolated, digested with Bam HI to release its 16-kb insert, and confirmed by means of dot blot and Southern hybridization. The positive clone was subcloned in pBluescript SK+ vector, sequenced, and characterized.
Sequence analysis revealed a full-length genomic clone with an open reading frame starting with an initiation codon (ATG) at position 1 and ending with a termination codon (TGA) at position 622. One putative polyadenylation signal (AATAAA) is identified at positions 951 in the 3' untranslated region, and 6 additional stop codons are located at positions 628, 769, 901, 946, 967, and 982 downstream from the start codon. In the 5' promoter region, a putative TATA box (TATTATTA) is located at position -72 upstream from the start codon. The deduced amino acid sequence has 207 residues and includes a putative signal peptide of 21 residues.
The results reveal for the first time information on the structure of a major peanut allergen, Ara h 2. Comparison of the cDNA and genomic sequences revealed the absence of an intron but the presence of 2 isoforms of Ara h 2 or different members of the same gene family.
花生已被确认为强效食物过敏原，可在过敏个体中引发危及生命的IgE反应。随着目前花生过敏情况的增加，迫切需要从分子层面表征编码目标蛋白的基因，并了解其调控本质。
本研究的目的是分离、测序并表征至少一个编码主要花生过敏原Ara h 2的全长基因组克隆。
用基于已发表的Ara h 2 cDNA部分序列5'端构建的80 bp寡核苷酸探针筛选构建于Lambda Fix II载体中的花生基因组文库。分离出一个推定的阳性λ克隆，用Bam HI酶切以释放其16 kb的插入片段，并通过斑点印迹和Southern杂交进行确认。将阳性克隆亚克隆到pBluescript SK +载体中，进行测序和表征。
序列分析揭示了一个全长基因组克隆，其开放阅读框从第1位的起始密码子（ATG）开始，到第622位的终止密码子（TGA）结束。在3'非翻译区的第951位鉴定出一个推定的聚腺苷酸化信号（AATAAA），在起始密码子下游的第628、769、901、946、967和982位还存在另外6个终止密码子。在5'启动子区域，一个推定的TATA框（TATTATTA）位于起始密码子上游-72位。推导的氨基酸序列有207个残基，包括一个21个残基的推定信号肽。
结果首次揭示了主要花生过敏原Ara h 2的结构信息。cDNA和基因组序列的比较显示不存在内含子，但存在Ara h 2的2种异构体或同一基因家族的不同成员。
