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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 ,为进一步研究NDRG1的功能打下了良好的基础
NDRG1 is an abnormally high expression of a new gene found in N-myc-deficient mouse embryonic tissue.In the study of the mechanism of atherosclerosis caused by hyperhomocysteinemia, in cultured human umbilical vein endothelial cells ( HUVEC) .In order to investigate the function of human NDRG1 and the relationship between structure and function, NDRG1 cDNA was cloned from total RNA of human brain by RT PCR and sequenced. NDRG1 was inserted into pPROEXHTb expression vector, E.coli DH5α competent cells were transformed and highly efficient and soluble expression was obtained in LB medium.The expressed 6His NDRG1 fusion protein was purified by Ni2 + NTA coupled agarose beads and its secondary structure was analyzed by circular dichroism, and the result : Α helix: 236, β sheet: 186, angle of rotation: 257, random coil: 32 0. The rabbit was immunized with the expressed protein to prepare a high titer antibody, The purification of antiserum by NDRG1 antigen on nitrocellulose membrane increased the specificity of the antibody, laying a good foundation for further study on the function of NDRG1