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克隆、表达和纯化重阳木花粉相关变应原profilin基因,并对其免疫学活性进行鉴定。采用RT-PCR和3-’RACE技术获得整个profilin基因的开放阅读框,将其与PET28a载体连接并转化大肠杆菌E.cdiBL21(DE3)进行诱导表达,通过Ni2+亲和层析柱对重组蛋白进行纯化,采用Western blot检测其IgE结合活性。克隆获得了profilin的全长基因,开放阅读框为396个碱基(包括终止密码子),编码131个氨基酸。成功地构建了原核表达载体,并在大肠杆菌中大量地表达了profi-lin,纯化后的重组蛋白进行免疫印迹,结果显示重阳木花粉过敏患者血清对重组profilin反应呈阳性。
The profilin gene was cloned, expressed and purified, and its immunological activity was identified. The entire open reading frame of profilin gene was obtained by RT-PCR and 3-’RACE. The recombinant protein was linked to PET28a vector and transformed into E. coli E.cdiBL21 (DE3) for expression. The recombinant protein was analyzed by Ni2 + affinity chromatography Purified and the IgE binding activity was detected by Western blot. The full-length gene of profilin was cloned and the open reading frame was 396 bases (including the stop codon), encoding 131 amino acids. The prokaryotic expression vector was successfully constructed and profi-lin was abundantly expressed in E. coli. The purified recombinant protein was immunoblotted and the results showed that the serum of patients with allergic reaction was positive for recombinant profilin.