采用亲和层析结合制备凝胶电泳获得高纯度的重组抗原蛋白用于制备蛋白质芯片

为了得到制备抗原芯片所需的高纯度重组抗原蛋白，需要建立一套适合于多种重组抗原表达和纯化的技术路线．采用了亲和层析结合制备胶电泳的方法，对16种用于构建蛋白质芯片的食管癌相关抗原基因进行了克隆重组并在大肠杆菌中进行了表达．对高表达的重组蛋白首先制备包涵体，然后采用Ni-Sepharose亲和层析得到初步纯化的蛋白质，最后使用SDS-PAGE制备胶电泳作进一步纯化．经过透析复性后，用于制备蛋白质芯片．采用亲和层析纯化重组蛋白，得率为71% ，纯度约为70%；在SDS-PAGE制备胶进一步纯化后，得率为32%，纯度为95%，经过透析和复性后，最终得率为21%，纯度为95%．得到的重组蛋白RPS4在ELISA检测中可以和血清中识别RPS4 的自身抗体起反应，并且，采用精纯抗原制备的蛋白质芯片，在检测抗原与抗体这一对反应中也具有较高的敏感性和特异性，适合大规模血清抗体的检测．研究表明，采用亲和层析结合制备凝胶电泳纯化抗原蛋白，是一条简便快捷，适合需要量不大，但对纯度要求比较高的蛋白质芯片制备的技术路线．

Efficient Purification of Tumor Antigen Proteins by Affinity Chromatograph and Prepared Gel Electrophoresis for Construction of Tumor Antigen Microarray

A parallelled purification procedure suitable for acquiring high purified multiple recombinant antigen proteins should be established for construction of antigen protein microarray. An efficient approach for purification of tumor antigen proteins by affinity chromatograph and prepared gel electrophoresis for construction of tumor antigen microarray was established and evaluated. In the procedure, the inclusion bodies were prepared firstly. Then, a Ni-NTA His-bind resins was applied to all dissolved inclusions. The yield and purity of the affinity purified step were 71% and 70% respectively. After the initiative affinity chromatograph purification, the eluted purified recombinant antigen proteins were runned on the prepared scaled SDS-PAGE, and the purified recombinant antigen protein bands were indicated by KCl solution and cutted out. Furthermore, a common dialysis and refolding procedure were applied to the electro-eluted purified recombinant antigen protein solutions. The yield and purity of this further purified step were 32% and 95% respectively. Furthermore, the reaction of purified recombinant RPS4 antigen against the anti-RPS4 autoantibody in esophageal cancer patients' sera was confirmed by ELISA. The 16 purified antigen proteins were pin-printed on the aldehyde glass slides for construction a protein microarray. The reaction of the RPS4 antigen in the microarray against the anti-RPS4 autoantibody in esophageal cancer patients' sera was the same as that assayed in ELISA. The research suggests that affinity chromatograph combining prepared gel electrophoresis was an efficient parallelled purification approach for tumor antigens in construction of microarray.