桃过敏原组分检测蛋白质芯片的制备及验证

Preparation and identification of protein chip for detecting components of peach allergen

目的制备用于检测血清中针对几种桃过敏原组分抗体的蛋白质芯片,为针对桃过敏原组分过敏患者的临床诊断提供快速可靠的方法。方法合成6种常见的桃过敏原组分的编码基因,分别将其插入p GAPZαA酵母表达载体,AvrⅡ单酶切线性化后,电转毕赤酵母SMD1168,表达和纯化相应的桃过敏原组分蛋白,并制备蛋白质芯片;用蛋白芯片检测临床血清样本中针对桃过敏原组分蛋白的抗体,与ImmunoCAP法相比,验证蛋白质芯片检测的灵敏度和特异性。结果用酵母表达系统成功表达并纯化了4种桃过敏原组分Pru p 1、Pru p 3、Pru p 4和Pru p 7,制备了能进行质量控制的检测血清桃过敏原组分抗体的蛋白质芯片。检测了疑似桃过敏患者血清41例,结果以ImmunoCAP法为金标准,蛋白芯片检测血清Pru p 1、Pru p 3和Pru p 4抗体的灵敏度分别为40%、100%和100%,特异性为78%、50%和100%;对上述3种组分抗体的综合检测灵敏度为86%,特异性为96%。针对缺乏ImmunoCAP检测结果的Pru p 7抗体,蛋白芯片检测出6份阳性的血清样本,均为桃过敏患者。结论桃过敏原组分检测芯片在灵敏度和特异性方面与ImmunoCAP相当,血清用量少,优化后有望成为临床桃过敏检测的一种新方法。

Objective To prepare a protein chip for the detection of serum antibodies against several peach allergen components,and to provide a rapid and reliable method for clinical diagnosis of peach allergic disease.Methods The coding sequences of six key peach components Pru p 1,Pru p 2,Pru p 3,Pru p 4,Pru p 7 and Pru p G were inserted into pGAPZα A yeast expression vector.Then the vectors were digested by endonuclease Avr Ⅱ and electroporated into yeast SMD1168 for protein expression.After peach proteins were purified,the protein chip was prepared and used to detect the antibodies against peach allergen components in serum samples from 41 suspected patients.The sensitivity and specificity of the protein chip were verified by comparison with ImmunoCAP method.Results The protein chip was prepared with these proteins for detecting Ig E antibodies in serum samples againstthese four peach allergen components.The results showed that the sensitivity of the protein chip to Pru p 1,Pru p 3 and Pru p 4 was 40%,100% and 100%,and the specificity was 100%,50% and 100% respectively.The total sensitivity and specificity was 86% and 96% respectively.Six serum samples from peach allergy patients were identified Pru p7 antibody positive by the protein chip.Conclusions The sensitivity and specificity of the protein chip is comparable to ImmunoCAP.It needs less serum and so to be a potential method for the clinical diagnosis of peach allergenic disease after optimization.