ZZ domain和APEX2融合蛋白的制备和应用

Preparation and application of ZZ domain and APEX2 fusion protein

目的基于免疫球蛋白结合结构域ZZ domain和过氧化物酶APEX2的融合表达,开发一种可以特异性结合免疫球蛋白且具有过氧化氢酶催化活性的新型免疫亲和检测试剂。方法将ZZ domain和APEX2融合表达蛋白——ZZ-APEX2在BL21(DE3)大肠杆菌进行大量表达,并利用IBA公司的strep-Tactin纯化系统进行纯化。通过点印迹和Western blotting检测ZZ-APEX2催化过氧化氢分解并启动鲁米诺发光反应的能力。通过愈创木酚比色法测定ZZ-APEX2的过氧化氢酶活性。结果ZZ-APEX2可以在大肠杆菌中可溶性表达,而且可以通过strep-Tactin系统纯化获取。ZZ-APEX2在室温保存48 h和在-20℃保存2个月均保持着稳定的过氧化氢酶活性,并可以用于Western blotting实验。通过双倒数法作图发现,与常规羊抗兔IgG-HRP和羊抗鼠IgG-HRP二抗相比,ZZ-APEX2具有更大的Kcat(s;)值。结论成功开发了一种分子量小、特异性好、容易大量生产、无需实验动物和繁琐的HRP蛋白提纯步骤的新型免疫亲和检测试剂。

This study was designed to prepare a new immunodetection reagent with immunoglobulinrecognition and hydrogen peroxide catalysis activity by combining ZZ domain and APEX2.ZZ domain and APEX2ORF were cloned into pRSETA expression plasmid,and the fusion protein ZZ-APEX2 was expressed by E.coli BL21(DE3)system.ZZ-APEX2 was tagged with strep at N-terminal for purification using Strep-Tactin purification system.Dotblot and Western blot were used to evaluate the ability of ZZ-APEX2 to degrade hydrogen peroxide and trigger chemiluminescent reaction.Furthermore,guaiacol colorimetry was used to determine the hydrogen peroxide enzyme activity of ZZ-APEX2.Data showed that most ZZ-APEX2 expressed in BL21(DE3)was kept in noninclusion status,and massive ZZ-APEX2 fusion protein was purified by Strep-Tactin purification system successfully.It was demonstrated that ZZ-APEX2 kept stable hydrogen peroxide catalysis activity both at room temperature for 48 hours and in-20℃condition for 2 months.It was also showed that ZZ-APEX2 was successfully applied in Western blotting assay.Furthermore,the result of Lineweaver-Burk plot confirmed that Kcat(s;)of ZZAPEX2 is higher than those of conventional HRP-conjugated secondary antibodies.In this study,a new immunodetection reagent with the advantage of small molecular mass,high specificity and easy production in scale has been developed.This immunodetection reagent avoids the large consumption of experimental animals and the complex purification process for HRP protein whichhappened in the preparation of conventionalimmunoglobulin-HRP-conjugated secondary antibody.