肿瘤转移抑制基因nm23-h1融合蛋白的构建、纯化 - 第三军医大学学报.doc

肿瘤转移抑制基因Nm23-H1融合蛋白的表达纯化及其功能的初步研究 张志敏*，杨雪琴*，王 东，戴 楠，曾林立，李增鹏，王 阁 （400042 重庆第三军医大学大坪医院野战外科研究所肿瘤中心） 利用人Nm23-H1基因原核表达载体，在大肠杆菌中表达并纯化融合蛋白，并对蛋白其活性进行初步分析。方法将重组质粒pET28a-Nm23-H1转化入E.coli BL21(DE3)，IPTG诱导表达并鉴定。用金属螯合层析技术进行蛋白纯化，得到Nm23-H1融合蛋白，用SDS及Western blot鉴定纯化蛋白，并用RP-HPLC法、电泳迁移率变动分析实验检测Nm23-H1融合蛋白的NDPK活性及AP修复活性。结果转化溶原菌后能够表达目的蛋白，蛋白表达量高，为可溶性蛋白。Ni2+柱纯化后得到Nm23-H1融合蛋白经SDS及Western blot鉴定正确。RP-HPLC法就及电泳迁移率变动分析实验证明所纯化的Nm23-H1融合蛋白具有NDPK活性，不具有AP修复活性，但可以增加APE1蛋白的AP修复活性。结论利用原核表达载体pET28a-Nm23-H1在E.coli BL21(DE3)中高效表达和纯化得到具有NDPK活性，但无AP修复活性的Nm23-H1融合蛋白，此蛋白可以增强APE1蛋白的AP修复活性共同参与细胞的DNA损伤修复。Nm23-H1基因；原核表达；蛋白纯化 Purification and functional characterization of Nm23-H1 fusion protein Zhang Zhimin*，Yang Xueqin*，Wang Dong，Dai Nan，Zeng Linli，Li Zengpeng，Wang Ge（Cancer Center， Research Institute of Surgery， Daping Hospital，Third Military Medical University ， Chongqing 400042，China） [Abstract] ObjectiveTo construct a prokaryotic expression vector for human Nm23-H1 genes，purify its fusion protein and study its activity. MethodsThe construction pET28a-Nm23-H1 was transformed into E.coli BL21 (DE3) and induced to express Nm23-H1 fusion protein with IPTG. The expressed Nm23-H1 fusion protein was identified by SDSand Western blot， and purified by affinity chromatographic with Ni2+ column. Immunogenicity and activity of Nm23-H1 fusion protein was analyzed by Western blot and EMSA. Results The prokaryotic expression vector of pET28a- Nm23-H1 vector could express Nm23-H1 fusion protein well in E.coli BL21 (DE3). The Nm23-H1 fusion protein， which has NDPK activity， could be purified via affinity chromatographic with Ni2+ column. But AP endonuclease activity of Nm23-H1 protein was too weak to detect. Conclusion The Nm23-H1 fusion protein is high expressed in E.coli BL21 (DE3) and successfully purified， which is significant for elucidating Nm23-H1’s biologic character in the future study. [Key words] Nm23-H1；prokaryotic expression；protein purificationSupported by the National Natural Science Foundation of Chi