背景:胡杨花粉是我国最常见的致敏花粉之一。然而,关于Hu草花粉中的致敏成分几乎没有发现。我们的研究旨在纯化和鉴定Hu草花粉的发病相关1(PR-1)蛋白,并表征这种新型过敏原的分子和免疫化学特性。
方法:采用色谱组合策略,从Hu草花粉提取物中纯化出天然PR-1蛋白(命名为HumjPR-1),并通过质谱鉴定。通过cDNA克隆证实了HumjPR-1的编码序列。从大肠杆菌中表达并纯化重组HumjPR-1。通过免疫印迹评估变应原性,酶联免疫吸附测定(ELISA),抑制ELISA,和嗜碱性粒细胞活化试验使用Humulus日本过敏患者的全血。通过计算机模拟方法对其理化性质和三维结构进行了全面表征。
结果:变应原性分析显示,76.6%(23/30)的Humusjaponicus花粉过敏患者表现出对天然HumjPR-1的特异性IgE识别。HumjPR-1的cDNA序列具有编码171个氨基酸的516bp开放阅读框。理化分析表明HumjPR-1是一种稳定且相对热稳定的蛋白质。HumjPR-1与其他同源过敏原共享类似的三维折叠模式,这是一个独特的αβα夹心结构,包含4个α-螺旋和6个反平行β-折叠,包含4个保守的CAP域。
结论:天然PR-1首先被纯化,并被鉴定为Humulusjaponicus花粉中的主要过敏原。这些发现将有助于开发Humus日本花粉症的诊断和治疗策略。
BACKGROUND: The
Humulus japonicus pollen is one of the most common allergenic pollens in
China. However, little is unveiled regarding the allergenic components in
Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from
Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen.
METHODS: The natural PR-1 protein (named as Hum j PR-1) was purified from
Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients\' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods.
RESULTS: The allergenicity analysis revealed that 76.6 % (23/30) of the
Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αβα sandwich structure containing 4 α-helices and 6 antiparallel β-sheets, encompassing 4 conserved CAP domain.
CONCLUSIONS: The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.