Cheong N, Yang L, Lee B W, Chua K Y
Bioprocessing Technology Center, Faculty of Medicine, National University of Singapore, Republic of Singapore.
Allergy. 2003 Apr;58(4):352-6. doi: 10.1034/j.1398-9995.2003.00033.x.
Blomia tropicalis is an important mite species in the tropics and subtropical regions of the world. It is well established that the allergen from this species of mite is one of the triggering factors for allergic asthma. The isolation and characterization of allergens in this mite species is desired to provide sensitive and specific reagents for diagnostic as well as therapeutic purposes.
The SMART (Clontech Laboratories, Palo Alto, CA, USA) rapid amplification of complementary DNA ends (RACE cDNA amplification) method was used to isolate the putative Blo t 3 gene. Polymerase chain reactions (PCR) were performed in the presence of specific gene primers to obtain the full-length gene, and were confirmed by DNA sequencing. The putative gene was cloned into E. coli expression vector GST-4T-1 and expressed as a fusion protein with glutathione-S-transferase (GST). The allergenicity of the GST-Blo t 3 recombinant protein was evaluated by human IgE enzyme-linked immunoassay (ELISA) and skin pricks tests.
The full length Blo t 3 gene had 1138 base pairs, including a 105-bp long 5' nontranslated region, an ATG start codon at positions 106-108, and a stop codon TAA at positions 904-906, with an open reading frame coding for a polypeptide of 266 amino acids. Protein analysis revealed that it was a serine protease that had a prepro-mature structure that shared high sequence homology with group 3 dust mite allergens. The predicted molecular weight of the matured protein was approximately 23.8 kD with a theoretical pI of 8.87. The frequency of IgE reactivity of the recombinant protein showed up to 50% of IgE reactivity with mite allergic subjects but IgE titer was generally low.
We had isolated and fully characterized the cDNA encoding an important B. tropicalis allergen that was highly homologous to Group 3 dust mite allergens and we proposed that it should be designated as Blo t 3. Its clinical importance was implicated by the high frequency of IgE reactivity with allergic sera.
热带无爪螨是世界热带和亚热带地区一种重要的螨类物种。已知该螨类物种的变应原是过敏性哮喘的触发因素之一。为了提供用于诊断和治疗目的的灵敏且特异的试剂，需要对这种螨类物种中的变应原进行分离和鉴定。
采用SMART（美国加利福尼亚州帕洛阿尔托的Clontech Laboratories公司）快速扩增互补DNA末端（RACE cDNA扩增）方法分离推定的Blo t 3基因。在特异性基因引物存在的情况下进行聚合酶链反应（PCR）以获得全长基因，并通过DNA测序进行确认。将推定基因克隆到大肠杆菌表达载体GST - 4T - 1中，并表达为与谷胱甘肽 - S - 转移酶（GST）的融合蛋白。通过人IgE酶联免疫吸附测定（ELISA）和皮肤点刺试验评估GST - Blo t 3重组蛋白的变应原性。
全长Blo t 3基因有1138个碱基对，包括一个105 bp长的5'非翻译区，在第106 - 108位有一个ATG起始密码子，在第904 - 906位有一个终止密码子TAA，其开放阅读框编码一个266个氨基酸的多肽。蛋白质分析表明它是一种丝氨酸蛋白酶，具有前原成熟结构，与3组尘螨变应原具有高度序列同源性。成熟蛋白的预测分子量约为23.8 kD，理论pI为8.87。重组蛋白的IgE反应频率显示，螨过敏受试者的IgE反应率高达50%，但IgE滴度通常较低。
我们已经分离并全面鉴定了编码一种重要的热带无爪螨变应原的cDNA，该变应原与3组尘螨变应原高度同源，我们建议将其命名为Blo t 3。其与过敏血清的IgE反应频率较高，提示了其临床重要性。
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