链亲和素-补体3d(SA-C3d)融合蛋白的制备及其功能检测  

Preparation of streptavidin-C3d fusion protein and detection of its function in vitro

作　　者：童玲 左安欣 牛婷婷 周世荣 高基民[1] TONG Ling;ZUO Anxin;NIU Tingting;ZHOU Shirong;GAO Jimin(School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]温州医科大学检验医学院,生命科学学院,浙江温州325035

出　　处：《细胞与分子免疫学杂志》2018年第7期583-588,共6页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的原核表达链亲和素-补体3d(SA-C3d)融合蛋白,体外探索蛋白活性。方法以C3 c DNA为模板扩增出C3d段DNA,使用一步克隆法将C3d段与酶切后的质粒p ET-24a-6His-SA-IL15相连,获得SA-C3d原核表达质粒。将测序正确的质粒转化入表达感受态大肠杆菌Rosetta中,诱导蛋白表达。使用镍柱亲和层析及梯度逐级减低的变性剂脲素透析复性的方法获得目的蛋白。通过锚定生物素化的MB49细胞实验反映SA的功能,通过促进Raji细胞生长的实验检测C3d的功能。结果成功构建SA-C3d原核表达质粒,通过镍柱纯化与透析复性获得高纯度目的蛋白。该蛋白特异性的与生物素化的MB49细胞结合,促进Raji细胞增殖并呈现剂量依赖效应,体现蛋白SA-C3d具有双功能活性。结论成功制备的SA-C3d融合蛋白可在体外与生物素化的MB49细胞结合并促进Raji细胞增殖。Objective To detect the prokaryotic expression of streptavidin-complement 3d( SA-C3d) fusion protein and verify its function in vitro. Methods The C3d DNA was amplified using C3c DNA as a template,and the C3d fragment was ligated with the vector plasmid p ET-24 a-6 His-SA-IL15 after the digestion with a one-step cloning method to obtain the SA-C3d prokaryotic expression plasmid. The correctly sequenced plasmid was transformed into expression competent Rosetta to induce protein expression. The target protein was obtained by nickel column affinity chromatography and urea dialyzed refolding. The function of SA was demonstrated by anchoring the biotinylated MB49 cel experiment,and the function of C3d was detected by an experiment that promoted the growth of Raji cells. Results The prokaryotic expression vector of SA-C3d was successfully constructed. The purified target protein was obtained by nickel column purification and dialysis refolding. The protein was specifically bound to biotinylated MB49 cells,which promoted the proliferation of Raji cells in a dose-dependent manner,indicating that the protein SA-C3d had a bifunctional activity. Conclusion The successfully prepared SA-C3d fusion protein can be bound to biotinylated MB49 cells in vitro and promote Raji cell proliferation.

关 键 词：链亲和素-补体3d(SA-C3d) 包涵体 原核表达 MB49细胞 

分 类 号：R392.1[医药卫生—免疫学] R392-33[医药卫生—基础医学]