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目的克隆、表达和鉴定青蒿花粉变应原Arta1。方法在成功构建青蒿花粉cDNA文库的基础上,用蒿属花粉过敏患者的阳性混合血清进行免疫学筛选,所获阳性克隆亚克隆入pET24a(+),经IPTG诱导表达后,通过Ni2+亲和层析柱对重组变应原进行纯化,并采用Westernblot和ELISA检测其IgE结合活性。结果选用阳性血清从青蒿花粉cDNA文库中筛选到1个阳性克隆,经序列测定,该基因与GenBank中已知基因无明显同源性,含有长度为609bp的开放阅读框,编码203个氨基酸,命名为Arta1;该重组变应原在大肠杆菌中高效表达为相对分子质量(Mr)为22.7×103蛋白,进一步在Ni2+亲和层析柱得到高度纯化;免疫学分析表明重组变应原有良好的IgE结合活性。结论本研究克隆和鉴定了一个青蒿花粉主要变应原Arta1(登录号为:CK700713),为花粉过敏性疾病的诊断和免疫治疗及进一步的实验研究奠定基础。
Objective To clone, express and identify A. annua pollen allergen Arta1. Methods Based on the successful construction of A. annua pollen cDNA library, the positive clones were screened by immunoblotting with the positive serum of Artemisia pollen allergy. The positive clones were subcloned into pET24a (+) and induced by IPTG. The positive clones were identified by Ni2 + affinity The column was used to purify the recombinant allergen, and its IgE binding activity was detected by Western blot and ELISA. Results A positive clone was screened from the cDNA library of Artemisia annua L. by using positive serum. The positive clones were sequenced and found to have no obvious homology with the known genes in GenBank. The gene contained a 609bp open reading frame (ORF) encoding 203 amino acids, Named as Arta1. The recombinant allergen was highly expressed in E. coli with a relative molecular weight of 22.7 × 103 and was highly purified on Ni2 + affinity chromatography. Immunological analysis showed that the recombinant allergen was good IgE binding activity. Conclusion This study cloned and identified Arta1, a major allergen of Artemisia annua pollen (accession number: CK700713), which laid the foundation for the diagnosis and immunotherapy of pollen allergic diseases and further experimental studies.