芒果果实中泛变应原profilin的克隆、表达及免疫学活性鉴定

目的　克隆并表达芒果果实中泛变应原肌动蛋白抑制蛋白(profilin)并了解其免疫学活性。方法利用RT-PCR结合:RACE技术克隆芒果果实中泛变应原profilin的全长基因,并进行序列分析。然后设计带有酶切位点的特异性引物,采用RT-PCR获得整个芒果profilin的开放阅读框,将其与pET-28a载体连接并转化大肠杆菌Escherichia coli BL21(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。结果克隆获得了芒果profilin的2个型。每个型的cDNA都包括一个编码131个氨基酸的开放阅读框。序列分析结果显示所克隆到的基因与许多水果和花粉的泛变应原profilin基因有很高的同源性(>70%)。根据变应原的命名规则,将它们分别命名为Man i 3.01和Man i 3.02,并将这两个基因提交到GenBank数据库中,其登录号分别为DQ270547和DQ400579。重组芒果profilin在大肠杆菌中高效的表达,进一步经Ni2 亲和层析柱纯化后经Western blot检测具有良好的免疫学活性。结论成功地克隆和表达了芒果profilin,并证明与芒果过敏者血清有特异IgE应答。

Cloning, expression and immunocharacterization of pan-allergen profilin in mango fruit (Mangifera indica L. )

Objective To clone, express pan-allergen profilin from mango fruit ( Mangifera indica) and to explore its immuno-activity. Methods RT-PCR and RACE methods were applied to clone the full-length pan-allergen genes from mango fruit and the sequences were analyzed. The specific primers were designed. The ORF of profilin of mango was amplified with RT-PCR and cloned into the expression vector pET-28a. Expression of the recombinant mango profilin was carried out in Escherichia coli BL21 (DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2 coupled to Sepharose. IgE reactivity of recombinant mango profilin was investigated by immunoblot. Results Two isoforms of mango fruits profilin were cloned. Each mango profilin cDNA includes an open reading frame coding for 131 amino acids. Sequence analysis showed that the two genes shared high identities( > 70%)with pan-allergen profilin from many fruits and pollen. So the clones were regarded as pan-allergen gene. The deduced proteins were named Man i 3.01 and Man i 3.02, respectively. The GenBank accession number of the clones was DQ270547 and DQ400579. After overexpressed in Escherichia coli BL21(DE3) , the recombinant protein was purified through affinity chromatography with Ni2 coupled to Sepharose . Immunoassay showed that the recombinant allergen has good IgE binding capacity. Conclusion The profilin of mango fruits was expressed successfully in BL21 (DE3) and showed IgE response with serum from patients being allergic to mango.