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cDNA cloning, expression and characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus

机译：烟曲霉变应原性L3核糖体蛋白的cDNA克隆，表达与鉴定

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Aspergillus fumigatus (Afu) is an important fungal pathogen causing allergic and invasive respiratory disorders. A plethora of multi-functional allergens/antigens secreted by Afu have been implicated in pathogenesis. The present study was undertaken to identify and characterize novel Afu allergen/antigen by cDNA library approach. cDNA library of Afu was immunoscreened with pooled sera of allergic bronchopulmonary aspergillosis (ABPA) patients. The cDNA clone, TS1, reacting significantly with specific IgG antibodies, was selected. cDNA was subcloned and expressed in Escherichia coli. Sequencing of the cDNA revealed an open reading frame (ORF) of 1179 bases coding for a protein with an approximate molecular weight of 44 kDa. Immunoreactivity of the recombinant TS1 protein (rTS1) was evaluated by ELISA and Western blot analysis using pooled sera of ABPA patients. The rTS1 exhibited binding to specific IgG and IgE antibodies present in sera of ABPA patients. The deduced amino acid sequence showed homology to 60S ribosomal protein L3 (RpL3) of Aspergillus nidulans, Saccharomyces cerevisiae and Homo sapiens. The RpL3 of S. cerevesiae, tcm1, to which TS1 sequence shows significant homology (72% identity), is known to be responsible for conferring resistance against trichodermin (antibiotic, inhibiting protein synthesis). The present study has led to identification, cloning and expression of a 44-kDa novel allergen/antigen of Afu with sequence homology to L3 ribosomal protein with a probable role in resistance of Afu to antifungal drugs. Sixty-four per cent sequence identity of Afu RpL3 with human RpL3 and common regions in their predicted epitopes suggest a possibility of involvement of Afu RpL3 in autoimmune reactions due to molecular mimicry.
机译：烟曲霉（Afu）是引起过敏性和侵入性呼吸系统疾病的重要真菌病原体。 Afu分泌的大量多功能过敏原/抗原与发病机理有关。本研究旨在通过cDNA文库方法鉴定和表征新型Afu过敏原/抗原。用过敏性支气管肺曲霉病（ABPA）患者的合并血清对Afu的cDNA文库进行免疫筛选。选择了与特异性IgG抗体显着反应的cDNA克隆TS1。 cDNA被亚克隆并在大肠杆菌中表达。 cDNA的测序显示一个开放阅读框（ORF）为1179个碱基，编码一个分子量约为44 kDa的蛋白质。重组TS1蛋白（rTS1）的免疫反应性通过ABPA患者合并血清的ELISA和Western blot分析进行评估。 rTS1显示出与ABPA患者血清中存在的特异性IgG和IgE抗体的结合。推导的氨基酸序列与构巢曲霉，酿酒酵母和智人的60S核糖体蛋白L3（RpL3）具有同源性。 TS1序列显示出显着的同源性（72％一致性）的酿酒酵母tcm1的RpL3已知可引起对曲霉毒素的抗性（抗生素，抑制蛋白质合成）。本研究已导致鉴定，克隆和表达了与L3核糖体蛋白具有序列同源性的Afu的44 kDa新型变应原/抗原，其可能在Afu对抗真菌药物的抗性中起作用。 Afu RpL3与人RpL3及其预测表位的共同区域有64％的序列同一性表明，由于分子模拟，Afu RpL3可能参与自身免疫反应。

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