神经元细胞信号传导蛋白质14-3-3ζ编码基因的扩增和克隆

Amplification and cloning of neuronal signaling protein 14-3-3ζ gene

目的 克隆神经元信号传导蛋白质 14 3 3ζ编码基因 ,以研究其在人神经退行性病变中的诊断价值、细胞信号传导过程中的作用及对细胞分裂、细胞凋亡的影响。方法 提取人脑胶质瘤细胞总mRNA ,以其为模板逆转录合成cD NA第一链 ,设计合成引物 ,用PCR扩增 14 3 3ζ基因编码序列 ,将其插入 pGEM T载体 ,并用双酶切和以质粒为模板的PCR进行鉴定。结果 RT PCR扩增出一条约 75 0bp大小的特异性条带 ,重组质粒的双酶切和以质粒为模板的PCR获得了一条与RT PCR大小相同的条带。经DNA测序证明所克隆的DNA片段与GenBank收录序列一致。结论 信号传导蛋白质 14 3 3ζ重组 pGEM T克隆载体的成功构建 ,为进一步研究提供了条件。

Objective To clone signaling protein 14 3 3ζ gene in order to study its signal transduction and evaluate its use as a marker for diagnosis of human spongiform encephalopthies including CJD. Methods Human cDNA first strand synthesis was driven by superscript ⅡRT using human brain tumor tissue as template. Specific primers were designed and synthesised. Coding region gene of 14 3 3ζ protein was amplified by RT PCR. The PCR product was cloned into pGEM T vector. The cloning vector (pGEM T 14 3 3) was identified by restriction analysis and PCR followed by DNA sequencing. Results A specific band about 750 bp was amplified in RT PCR.The insert was confirmed by double restriction digestion of recombinant plasmid and PCR using recombinant plasmid as template. Conclusion pGEM T 14 3 3 was successfully constructed which provided the basis for further study on signal protein 14 3 3 expression and its function.