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目的 用DNA重组方法去除胶质细胞源性神经营养因子 (GDNF)受体 (GFRα 1)基因 3’ 端的与细胞膜糖基化磷脂酰肌醇 (GPI)相连的信号序列 ,使表达的GFRα 1以可溶性形式介导GDNF的作用。 方法 设计一对引物 ,其中下游引物含有 6个组氨酸密码子序列 ,以人GFRα 1全长cDNA为模板 ,PCR扩增编码可溶性GFRα 1序列并亚克隆于pBK RSV真核表达载体 ,转染COS 7细胞 ,收获培养上清 ,进行SDS PAGE及Western免疫印迹分析 ;使用钴离子金属螯合层析柱进行纯化 ;检测RET酪氨酸磷酸化以鉴定其生物学活性。 结果 Western免疫印迹证实培养上清液中有分子量为 6 0kD的特异阳性条带 ,与糖基化的GFRα 1分子量相符 ;金属螯合层析得到纯度达90 %以上的重组GFRα 1蛋白 ,每mlCOS 7培养上清中含有 0 5 μg。 结论 带 6个组氨酸尾的重组可溶性GFRα 1能够介导GDNF的作用 ,可引发酪氨酸激酶RET的磷酸化
Objective To delete the signal sequence linked to the glycosylated phosphatidylinositol (GPI) at the 3 ’end of the glial cell line derived neurotrophic factor (GDNF) receptor (GFRα 1) gene by DNA recombination, so that the expressed GFRα 1 Soluble forms mediate the effects of GDNF. Methods A pair of primers was designed, in which the downstream primer contained 6 histidine codon sequences. The full-length cDNA of human GFRα 1 was used as template to amplify soluble GFRα 1 sequence by PCR and subcloned into pBK RSV eukaryotic expression vector. COS 7 cells were harvested and the culture supernatant was harvested for SDS PAGE and Western immunoblot analysis; purified using cobalt ion metal chelate chromatography; and RET tyrosine phosphorylation was detected to identify its biological activity. Results Western blotting confirmed that there was a specific positive band with a molecular weight of 60 kD in the culture supernatant, which was consistent with the molecular weight of glycosylated GFRα 1. The recombinant GFRα 1 protein with a purity of over 90% was obtained by metal chelation chromatography. 7 culture supernatant containing 0 5 μg. Conclusions Recombinant soluble GFRα 1 with 6 histidine tails mediates the effects of GDNF and triggers the phosphorylation of tyrosine kinase RET