三裂叶豚草花粉新致敏蛋白组分超氧歧化酶克隆表达与致敏性

Cloning,expression,and allergenicity of superoxide dismutase,a novel allergen of Ambrosia trifida pollen

目的克隆和表达三裂叶豚草花粉中的一种新致敏蛋白组分超氧歧化酶,探讨其在中国人群中的致敏特征。方法通过对三裂叶豚草花粉转录组学研究,鉴定到一种潜在的超氧歧化酶基因序列,对基因进行克隆,通过表达和纯化获得蛋白,用clustalX2将已报道的超氧歧化酶与该基因进行多重序列比对,通过酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和免疫印迹实验(Western blot)研究三裂叶豚草花粉超氧歧化酶的致敏性。结果三裂叶豚草花粉超氧歧化酶序列编码区含465个碱基,编码154个氨基酸。多重序列比对发现三裂叶豚草花粉超氧歧化酶与橄榄花粉过敏原Ole e 5氨基酸序列存在一定的保守性,为53.9%。ELISA结果显示三裂叶豚草花粉超氧歧化酶的IgE结合率为12%(5/42),5份阳性血清在免疫印迹实验中均显示阳性结合带。结论三裂叶豚草花粉超氧歧化酶是一种新的致敏蛋白组分,本研究对完善三裂叶豚草花粉过敏原组分信息具有重要意义,为三裂叶豚草花粉过敏疾病的组分解析诊断方法的建立奠定了基础。

Objective To investigate the cloning and expression of superoxide dismutase(SOD),a new allergen of Ambrosia trifida pollen and to evaluate its allergenicity among Chinese population.Methods The sequence of a potential allergen,SOD of Ambrosia trifida pollen was identified by using transcriptomics sequencing technique.Then,the gene of Ambrosia trifida pollen SOD(Amb t SOD)was cloned and protein was expressed and purified.Multiple sequence alignments of Amb t SOD versus reported SOD were performed by using clustalX2 software.The allergenicity of Amb t SOD was detected by using enzyme-linked immunosorbent assay(ELISA)and Western blot.Results There are 465 base pairs in coding region of Amb t SOD sequence,which encode 154 amino acids.Based on the results of multiple sequence alignments,the amino acid sequence of Amb t SOD showed considerable homology compared to olive pollen allergen Ole e 5(53.9%).The binding rate of Amb t SOD to IgE was 12%(5/42)which was verified by using ELISA,and all the 5 positive sera showed positive bands in Western blot hybridization.Conclusions Amb t SOD was confirmed to be a new allergen with high immunogenicity.Amb t SOD is of far reaching importance for perfecting the information of allergenic components of Ambrosia trifida pollen,providing the basis for component resolved diagnosis of Ambrosia trifida pollen-allergic diseases.