人NDRG1的融合表达、纯化及抗体制备  

Fusion Expression, Purification of Human NDRG1 and Antibody Preparation

作　　者：张健[1] 刘新平[1] 张伟[1] 吴国强[1] 沈岚[1] 王立峰[1] 苏金[1] 张万会[2] 药立波[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,西安710032 [2]第四军医大学基础部生理学教研室,西安710032

出　　处：《中国生物化学与分子生物学报》2003年第6期698-703,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金 (No 3 0 0 70 773和No 3 0 1710 44 )~~

摘　　要：NDRG1是在N myc缺陷的小鼠胚胎组织中发现的一异常高表达的新基因 .在研究高同型半胱氨酸血症引起动脉粥样硬化的机制时 ,在培养的人脐静脉内皮细胞 (HUVEC)中发现了人NDRG1 .为了研究人NDRG1的功能以及结构与功能之间的关系 ,用RT PCR技术 ,从人脑总RNA中克隆NDRG1cDNA ,进行序列测定后 ,将NDRG1插入pPROEXHTb表达载体中 ,转化E .coliDH5α感受态细胞 ,并在LB培养基中获得高效可溶表达 .表达的 6His NDRG1融合蛋白经Ni2 + NTA偶联的琼脂糖珠纯化 ,通过圆二色性分析其二级结构 ,结果为 :α螺旋 :2 3 6 ,β片层 :1 8 6 ,转角 :2 5 7,无规卷曲 :32 0 .用此融合表达的蛋白免疫家兔 ,制备得到高效价的抗体 ,利用固定于硝酸纤维素膜上的NDRG1抗原亲和吸附纯化抗血清提高了抗体的特异性 。NDRG1 is a new gene which expresses abnormally highly in N myc mutant mouse embryos. Human NDRG1 is isolated from human umbilical vein endothelial cells in a study of homocysteine promoting arteriosclerosis diseases. In order to investigate the function and structure function relationship, NDRG1 was expressed in E.coli and the fusion protein was obtained. The RT PCR was used to clone human NDRG1 cDNA from the total RNA of brain and then the gene was sequenced. The recombinant vector NDRG1 pP RO EX HTb was transformed into E.coli DH5, and the strains highly expressing soluble 6His NDRG1 in LB medium were obtained. The fusion protein was purified by Ni 2+ NTA agarose beads. The secondary structure of purified 6His NDRG1 fusion protein was analyzed by circular dichroism. The results indicated that 6His NDRG1 was composed of helix:23 6, β\|sheet:18 6,turn:25 7 and random:32 0. Immunization in rabbits with the fusion protein was used to obtain the high titer antibody against NDRG1. The specific antibody was purified by absorbing antiserum with NDRG1 antigen immobilized NC filter. These works lay a favorable foundation for the study of the NDRG1 structure and function.

关 键 词：融合表达 圆二色性 免疫血清 抗体制备 基因表达 NDRG1基因 

分 类 号：Q786[生物学—分子生物学]