G蛋白Rab3a协同神经生长抑制因子(GIF)抑制神经元的生长

为了研究G蛋白Rab3a与神经生长抑制因子 (growthinhibitoryfactor,GIF ,原称金属硫蛋白 3,MT 3)相互作用对神经元细胞生长的影响 ,以嗜铬细胞瘤株 (pheochromocytoma)PC1 2充当神经元模型 .将hMT 3(humanMT 3)和Rab3a基因分别克隆至真核表达载体pFlag CMV 2和pSV HA中 ,质粒共同转染PC1 2细胞 ,观察转染后细胞的生长状态 .以共转染pFlag CMV 2 hMT 1和pSV HA Rab3a的细胞组作为对照 ,验证hMT 3与Rab3a相互作用对PC1 2影响的特异性 .结果发现 ,共转染pFlag CMV 2 hMT 3和pSV HA Rab3a的PC1 2细胞生长明显受到抑制 ,细胞生长抑制率与GIF在脑提取物存在F的神经元生长抑制作用接近 ,但转染两基因中的任何单个基因以及共转染pFlag CMV 2 hMT 1和pSV HA Rab3a对PC1 2细胞生长无影响 .进一步构建重组表达质粒pGEX 4T 1 Rab3a和pGEX 4T 1 hMT 3,转化大肠杆菌BL2 1 ,经谷胱甘肽 Sepharose 4B亲和层析、凝血酶酶切和SephacrylS 1 0 0纯化 ,得到纯度 95 %以上的Rab3a和hMT 3蛋白 .体外细胞生物学活性检测表明 ,表达的Rab3a蛋白与重组hMT 3蛋白共培养PC1 2 ,对细胞的生长产生了明显的特异性协同抑制作用 ,抑制曲线与GIF在脑提取物存在下的神经元生长抑制曲线极为相似

The Cooperated Effect of G-Protein Rab3a and GIF to Inhibit Neuron Growth

To study the function of cooperated role of G protein Rab3a and the growth inhibitory factor(GIF,originally termed as MT 3),pheochromocytoma cell line PC12 was used as neuron model.The genes of human MT 3 and Rab3a were cloned into pFlag CMV 2 and pSV HA,respectively,and subsequently cotransfected into PC12 cells by a lipofectamine kit.Another pair of cloned genes of pSV HA Rab 3a and pFlag CMV 2 hMT 1 was also cotransfected into PC12 as control.After cultivation,the obvious growing status was only observed in the cloned group containing recombinant hMT 3,which demonstrated similar results to the neuron treated with GIF,but the control and the clone transfected only with pFlag CMV 2 or pSV HA had no remarkable effects.Expressionable recombinants were constructed by linking Rab3a and hMT 3 to pGEX 4T 1,which were then transfected into BL21,expressed,and at last purfied by affinity chromatography,thrombin digestion and gel filtration on Sephacryl S 100.The final products with high purity were used to co cultivate PC12,and the cellular biological activities were tested by in vitro experiment.The results confirm that they have specific cooperative repressng effect on PC12 cellular growth,and the corresponding inhibitory curve is similar to the neuron treated with GIF and brain extracts.