Li Qiong, Zhu Yue, Zhang Fu-Xian, Cheng Ya-Li, Yang Yong-Shi, Wei Ji-Fu, Sun Jin-Lyu, Xu Zhi-Qiang
Department of Pharmacy, The Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research, Nanjing, China.
Department of Allergy, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.
World Allergy Organ J. 2025 Aug 7;18(8):101104. doi: 10.1016/j.waojou.2025.101104. eCollection 2025 Aug.
(HJ) is widely spread in East Asia. HJ pollen is one of the common allergens in the north of China in autumn. However, some controversy exists regarding the major allergen Hum j 1 from HJ pollen, which hamper its application in the diagnosis and treatment of HJ pollen allergy. Further studies are urgently needed to improve this aspect.
During our work in isolating potential allergens from HJ pollen, we found a protein of around 10 kDa with obvious immunoglobulin E (IgE) binding activity than other fractions. This protein was then further identified by mass spectrometry, and its gene sequence was matched from our in-house built transcripts of HJ pollen based on the identified peptides. The gene was further confirmed by polymerase chain reaction (PCR) cloning, and the recombinant protein was prepared by () expression system. The circular dichroism (CD) spectrum of natural and recombinant proteins was analyzed. The allergenicity of its natural and recombinant forms was assessed in Chinese patients and the relationship between the epitopes and its structural features was investigated under reduced, denatured and heat-treated conditions.
The 10 kDa protein with obvious IgE binding activity was finally purified from HJ pollen with a purity of 98%. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) results identified 6 internal peptides, which covered 98.9% of the mature sequence of the 10 kDa protein with the N-terminal segment as DNCFENGMK. We surprisingly found that the 10 kDa protein was previously underrecognized Hum j 1 based on its limited sequence of only N-terminus. The sequence failed to be classified into any protein families by InterPro functional analysis, indicating the Hum j 1 could be the orphan protein. Both natural and recombinant Hum j 1 exhibited similar CD spectrum. The specific IgE binding rate against them was determined as 79% (11/14) in patients' serum with HJ pollen allergy by ELISA with a strong correlation in absorbance values (R = 0.99, < 0.0001) and decreased by 76%-87% after broking disulfide bonds, with no significant change after heat treatment or urea treatment.
These findings first provided solid evidence about the major allergen Hum j 1 in HJ pollen and resolved its unclear status. The defined sequence could enhance understanding of Hum j 1 properties and aid in producing high-quality recombinant allergens for diagnosing and treating HJ pollen allergy.
豚草（HJ）在东亚广泛分布。HJ花粉是中国北方秋季常见的变应原之一。然而，关于HJ花粉主要变应原Hum j 1存在一些争议，这阻碍了其在HJ花粉过敏诊断和治疗中的应用。迫切需要进一步研究来改善这一状况。
在我们从HJ花粉中分离潜在变应原的工作过程中，我们发现一种约10 kDa的蛋白质，其免疫球蛋白E（IgE）结合活性比其他组分明显更高。然后通过质谱对该蛋白质进行进一步鉴定，并根据鉴定出的肽段从我们内部构建的HJ花粉转录本中匹配其基因序列。通过聚合酶链反应（PCR）克隆进一步确认该基因，并通过（）表达系统制备重组蛋白。分析天然蛋白和重组蛋白的圆二色性（CD）光谱。在中国患者中评估其天然和重组形式的变应原性，并在还原、变性和热处理条件下研究表位与其结构特征之间的关系。
最终从HJ花粉中纯化出具有明显IgE结合活性的10 kDa蛋白质，纯度为98%。液相色谱 - 串联质谱（LC-MS/MS）结果鉴定出6个内部肽段，覆盖了10 kDa蛋白质成熟序列的98.9%，其N端片段为DNCFENGMK。我们惊讶地发现，基于其仅N端的有限序列，该10 kDa蛋白质以前未被充分认识为Hum j 1。通过InterPro功能分析，该序列未能归类到任何蛋白质家族，表明Hum j 1可能是孤儿蛋白。天然和重组的Hum j 1均表现出相似的CD光谱。通过ELISA测定，在HJ花粉过敏患者血清中针对它们的特异性IgE结合率为79%（11/14），吸光度值具有强相关性（R = 0.99，<0.0001），在二硫键断裂后降低76% - 87%，热处理或尿素处理后无显著变化。
这些发现首次为HJ花粉中的主要变应原Hum j 1提供了确凿证据，并解决了其不明确的地位。确定的序列可以增强对Hum j 1特性的理解，并有助于生产用于诊断和治疗HJ花粉过敏的高质量重组变应原。
