METHODS: The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test.
RESULTS: A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients\' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test.
CONCLUSIONS: Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy.
方法:通过三步色谱法纯化了悬铃木花粉中的天然磷酸丙糖异构酶,并通过质谱鉴定。该蛋白质的cDNA序列与基于内部肽序列的内部转录物匹配,通过PCR克隆进一步证实。从大肠杆菌中表达并纯化重组磷酸丙糖异构酶。通过酶联免疫吸附试验对该蛋白进行致敏性分析,免疫印迹,和嗜碱性粒细胞激活试验.
结果:首次在悬铃木花粉中鉴定出属于丙糖磷酸异构酶的新变应原群,命名为Plaa7。Pla一7的cDNA包含一个762bp的开放阅读框,编码253个氨基酸。通过ELISA,天然Plaa7对患者血清显示41.4%的IgE反应性,其中吸光度值与针对悬铃木花粉提取物的血清sIgE相关。在不同的Plaa7阳性血清中,IgE与花粉提取物结合的抑制作用达到26%-94%。重组Plaa7在ELISA中表现出比其天然形式更弱的IgE反应性,但在免疫印迹中显示出相当的活性。通过嗜碱性粒细胞活化试验进一步证实了变应原性。
结论:磷酸三糖异构酶(Plaa7)首先被认为是悬铃木花粉中的花粉过敏原,这是一种与以前报道的完全不同的花粉过敏原。这一发现对于丰富有关过敏原成分的信息并为悬铃木花粉过敏的分子诊断或治疗策略铺平道路至关重要。