青蒿花粉变应原Art a1基因的克隆、表达及特性鉴定

Cloning, expression and immunocharacterization of Artemisia apiacea Hance allergen, Art a1

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 characterize an Artemisia apiacea Hance allergen, Art al. Methods On the basis of successful construction of eDNA library of Artemisia apiacea Hance, we immunoscreened eDNA library with sera pool from patients allergic to Artemisia. Positive clone was ligated into pET24a （ ＋ ） for expression. After induced by IFIG, Recombinant protein was purified through metal （Ni2 ^＋ ） cheltaing affinity chromatography. Western blot and ELISA were used to determine the IgE-binding capacity of the recombinant allergen. Results A positive clone was isolated and named Art al, sequence analysis indicated that this gene had no homology to the other gene of GenBank, contained a 609 open reading fragment and encoded 203 amino acids. After overexpressed in E. coli, the Mr 22.7 × 10^3 recombinant protein was purified through one-step affinity chromatography. Immunoassay showed that the recombinant allergen has good lgE-binding capacity. Conclusion A recombinant Artemisia allergen Art al （Access number： CK700713）has been isolated and characterized, which could be a useful tool for further study on pollen related allergy.