Mora C, Flores I, Montealegre F, Díaz A
Department of Microbiology, University of Puerto Rico, Medical Sciences Campus, School of Medicine, San Juan, Puerto Rico.
Clin Exp Allergy. 2003 Jan;33(1):28-34. doi: 10.1046/j.1365-2222.2003.01480.x.
House dust mite allergens have been shown to be a very important stimulus in the causation of asthma and triggers for the exacerbation of symptoms. Therefore, characterization of mite-derived allergens at the molecular level is an important step for the development of effective diagnostic and therapeutic approaches, as well as for epidemiological studies.
To clone, express and characterize at the molecular level the cysteine protease from Blomia tropicalis (Bt).
A full-length cDNA encoding Blo t 1 was cloned from a Bt cDNA library using a PCR and RACE-based strategy. The cDNA was PCR-amplified, sequenced and subcloned into a prokaryotic expression vector. The allergenicity of the recombinant Blo t 1 was evaluated for IgE reactivity by Western blot.
Blo t 1 cDNA encodes a 221 amino acids polypeptide with an estimated molecular weight of 25 kDa. The recombinant protein is 35% identical to other mite cysteine proteases. Recombinant Blo t 1 (rBlo t 1) bound IgE from 62% of Bt skin test-positive serum. Dermatophagoides pteronyssinus (Dp) skin test-positive sera did not react with rBlo t 1, indicating the possible presence of unique IgE epitopes on the rBlo t 1 molecule. A three-dimensional image of Blo t 1, constructed based on predicted analysis, showed conserved secondary and tertiary structure with other cysteine proteases.
We report the cloning, expression and IgE reactivity of Blo t 1, a novel allergen from the domestic mite Blomia tropicalis (Bt), highly homologous to cysteine proteases. This putative cysteine protease, designated Blo t 1, may play a major role as an immunodominant allergen involved in dust mite-specific IgE-mediated hypersensitivity.
屋尘螨过敏原已被证明是哮喘发病的一个非常重要的刺激因素，也是症状加重的诱因。因此，在分子水平上对螨源过敏原进行表征是开发有效的诊断和治疗方法以及开展流行病学研究的重要一步。
克隆、表达热带无爪螨（Bt）的半胱氨酸蛋白酶并在分子水平上对其进行表征。
采用基于PCR和RACE的策略从Bt cDNA文库中克隆编码Blo t 1的全长cDNA。对该cDNA进行PCR扩增、测序，并亚克隆到原核表达载体中。通过蛋白质免疫印迹法评估重组Blo t 1的过敏原性，检测其与IgE的反应性。
Blo t 1 cDNA编码一个由221个氨基酸组成的多肽，估计分子量为25 kDa。该重组蛋白与其他螨半胱氨酸蛋白酶有35%的同源性。重组Blo t 1（rBlo t 1）与62%的Bt皮肤试验阳性血清中的IgE结合。粉尘螨（Dp）皮肤试验阳性血清与rBlo t 1无反应，表明rBlo t 1分子上可能存在独特的IgE表位。基于预测分析构建的Blo t 1三维图像显示，其与其他半胱氨酸蛋白酶具有保守的二级和三级结构。
我们报道了来自家螨热带无爪螨（Bt）的一种新型过敏原Blo t 1的克隆、表达及其与IgE的反应性，该过敏原与半胱氨酸蛋白酶高度同源。这种推定的半胱氨酸蛋白酶命名为Blo t 1，可能作为一种免疫显性过敏原，在尘螨特异性IgE介导的超敏反应中起主要作用。
