荔枝果实中profilin的表达纯化及其与桦树花粉Bet v 2的交叉反应性研究

目的 表达和纯化荔枝果实profilin,并对其免疫学活性及交叉反应性进行研究.方法采用RT-PCR获得整个荔枝profilin的开放阅读框,将其与PET28a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,并通过ELISA抑制实验对其与桦树花粉profilin的交叉反应性进行研究.结果成功的构建了原核表达载体,并在大肠杆菌中大量的表达了荔枝profilin,纯化后的重组蛋白进行免疫印迹,结果显示15个荔枝过敏患者中有5个存在重组pofilin的特异性IgE抗体.ELISA抑制实验发现它们之间存在很高的交叉反应性.结论 成功的表达了荔枝profilin,经纯化后具有很好的免疫学活性,为进一步的深入研究奠定了基础.

Expression and purification of profilin from lychee fruit and its cross-reactivity with birch pollen profilin Bet v 2

Objective To express and purify the profilin from lychee fruits and study on its immunological characteristics and cross-reactivity with other allergens. Methods The ORF of profilin from lychee was amplified with RT-PCR and cloned into the expression vector pET 28a. The recombinant lychee profilin was exoressed in Escherichia coli BL21(DE3) and the expressed protein was purified by affinity chromatography with Ni2 coupled to sepharose. IgE reactivity of recombinant lyhee profilin was investigated by immunoblotting. IgE-inhibition and ELISA inhibition experiments were performed to analyze lychee profilin cross-reactivity.Results The expression vector was constructed successfully. The target protein was highly expressed in BL21 (DE3). The results of immunoblotting showed that 5 out of 15 tested lychee-allergic patients presented specific IgE-antibodies to recombinant lychee profilin. ELISA inhibition experiments showed a high cross-reactivity between lychee profilin and birch profilin in sera from lychee allergic patients.Conclusion The profilin of lychee fruits is expressed successfully in BL21(DE3). The recombination protein is purified and has immunological characteristics, which lays the foundation for further deeply research.