[1]张志敏,杨雪琴,许文,等.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[J].第三军医大学学报,2010,32(17):1824-1827.
 Zhang Zhimin,Yang Xueqin,Xu Wen,et al.Purification of Nm23-H1 fusion protein expression and its function[J].J Third Mil Med Univ,2010,32(17):1824-1827.
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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(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
32卷
期数:
2010年第17期
页码:
1824-1827
栏目:
论著
出版日期:
2010-09-15

文章信息/Info

Title:
Purification of Nm23-H1 fusion protein expression and its function
作者:
张志敏杨雪琴许文戴楠曾林立李增鹏王东王阁
第三军医大学大坪医院野战外科研究所肿瘤中心;解放军第456医院肿瘤科
Author(s):
Zhang ZhiminYang XueqinXu WenDai NanZeng LinliLi ZengpengWang DongWang Ge
Cancer Center,Institute of Surgery Research,Daping Hospital,Third Military Medical University,Chongqing, 400042,Department of Oncology, No. 456 Hospital of PLA,Jinan, Shandong,250031, China
关键词:
Nm23-H1基因原核表达蛋白纯化
Keywords:
Nm23-H1 geneprokaryotic expressionprotein purification
分类号:
R394-33; R394.3; R784
文献标志码:
A
摘要:
目的    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-PAGE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融合蛋白的核苷二磷酸激酶(NDPK)活性及核酸内切酶(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-PAGE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。
Abstract:
Objective To construct the prokaryotic expression vector for human Nm23-H1 genes and purify its fusion protein and study its activity. Methods Recombinant plasmid pET28a-Nm23-H1 was transformed into E.coli BL21 (DE3) and its expression was induced and identified with IPTG. The expressed Nm23-H1 fusion protein was purified by affinity chromatographic with Ni2+ column and identified by SDS-PAGE and Western blotting. Immunogenicity and activity of Nm23-H1 fusion protein were analyzed by RP-HPLC and EMSA. Results The prokaryotic expression vector of pET28a-Nm23-H1 could express the Nm23-H1 fusion protein in E.coli BL21 (DE3). The Nm23-H1 fusion protein with NDPK activity could be purified by affinity chromatographic with Ni2+ column. However, the AP endonuclease activity of Nm23-H1 protein was too weak to be detected. Conclusion Nm23-H1 fusion protein is highly expressed in E.coli BL21 (DE3) and can be purified,which is of great significance for elucidating the biologic characteristics of Nm23-H1 genes.

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更新日期/Last Update: 2010-09-08