日本柳杉花粉变应原CJP-6的重组表达和免疫反应性鉴定

Expression and immunoreactivity of Cryptomeria japonica pollen allergen,CJP-6

目的:获得日本柳杉花粉主要变应原CJP-6的编码基因,构建其原核表达载体进行表达,并对重组CJP-6(rCJP-6)进行免疫活性鉴定。方法:从日本柳杉花粉中提取总RNA,采用RT-PCR的方法扩增CJP-6编码基因,将其克隆入pET-19b表达载体。转入大肠杆菌BL21 Star(DE3)pLysS,经IPTG诱导表达,以Ni2+亲和层析柱对重组变应原进行纯化,采用Dot blot和ELISA方法检测重组变应原rCJP-6与对日本柳杉花粉、尘螨和蒿草花粉过敏患者的血清中的IgE反应活性。结果:重组变应原与日本柳杉花粉过敏患者血清中的IgE具有较高的结合活性,rCJP-6与尘螨、蒿草花粉过敏患者血清中的IgE也具有很高的反应性,而且反应性与日本柳杉花粉过敏的患者血清相似。结论:制备并获得了具有IgE结合活性的重组日本柳杉花粉变应原CJP-6,日本柳杉花粉变应原是中国过敏反应性疾病患者潜在的变应原,为国内过敏反应性疾病的临床诊断和免疫治疗及进一步的实验研究奠定了基础。

Objective：To construct a recombinant prokaryotic expression vector to express Japanese cedar（Cryptomeria japonica） pollen allergen,CJP-6,and to identify IgE-binding capacity of the recombinant allergen.Methods：The total RNA was extracted from C.japonica pollen,and then the CJP-6 gene fragments were amplified by RT-PCR and cloned into vector pET-19b.The recombinant plasmids were transformed to E.coli BL21 Star（DE3） pLysS.After induced by IPTG,the recombinant protein was purified through Ni2＋ affinity chromatography.The IgE binding activity of rCJP-6 is assessed using sera from patients allergic to C.japonica pollen,mite and Artemisia pollen by Dot blot and ELISA.Results：The recombinant allergen was expressed in E.coli and purified through Ni^2＋ affinity chromatography successfully.The rCJP-6 has high affinity with IgE existing in sera of patients allergic to C.japonica pollen,mite and Artemisia pollen.Conclusion：The rCJP-6,which has high IgE-binding activity,was expressed and purified.It will be a practical tool for clinical detection,immunotherapy and further study for allergic diseases.