小鼠脑14-3-3蛋白ζ亚型cDNA克隆及其原核表达分析

14-3-3是一类脑中丰富存在的蛋白质。在许多神经疾病患者的脑脊液中含量升高。最近的研究显示该蛋白相关于Parkinson病的病理过程。本研究采用RT-PCR法从小鼠脑中分离出编码14-3-3ζ亚型的cDNA片段,构建了基因重组质粒,分析了14-3-3ζ亚型cDNA在原核细胞中的表达并制备了基因重组蛋白。结果显示,RT-PCR扩增产物为738bp,编码245个氨基酸。重组质粒在原核细胞中的表达受IPTG诱导呈时间依赖性递增。纯化的目的蛋白在SDS-PAGE中的表观分子量为32kD,在Su-perdexS-200HR中的表观分子量为96kD。每升菌液可收获目的蛋白4.0mg。研究结果提示,14-3-3ζ亚型cDNA在原核细胞中高水平表达,基因重组蛋白易纯化可用于制备抗体和研究其生理功能。

cDNA CLONING AND PROKARYOTIC EXPRESSION OF MOUSE BRAIN 14-3-3 ζ ISOFORM PROTEIN

14-3-3 proteins are abundantly expressed in the brain and have been detected in the cerebrospinal fluid of patients with different neurological disorders. The results of recent studies have suggested that the proteins are related to the pathological processes of the Parkinson’s disease. In the present study, we isolated a cDNA fragment encoding the 14-3-3 ζ isoform from mouse brain by RT-PCR method. We also investigated the expression of the isolated cDNA in prokaryotic cell, and purified recombinant 14-3-3 ζ protein. the isolated cDNA fragment was composed of 738 bp and encodes 245 amino acids. The fusion protein GST-m1433z was expressed in the bacteria, with IPTG present in the condition medium. The recombinant mouse 14-3-3 ζ was purified to homogeneity from soluble fraction of the bacteria. The molecular weight of the purified protein was calculated to be approximately 96 kD on Superdex S-200HR chromatography and to be 32 kD on SDS-PAGE in the presence of β-mercaptoethanol. Western blot analysis demonstrated that the purified recombinant protein was reactived to the anti 14-3-3 ζ monoclonal antibody. These results indiate that mouse 14-3-3 ζ cDNA is effectively expressed with bacteria, and pure recombinant mouse 14-3-3 ζ is prepared, which may be samples to study physiological function of the protein.