胶质细胞源性神经营养因子基因的克隆及其在大肠杆菌中的高效表达

Isolation of the gene encoding glial cell line derived neurotrophic factor and its high expression in E.coli

目的：分离胶质细胞源性神经营养因子（ｇｌｉａｌ ｃｅｌｌ ｌｉｎｅ－ｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ，ＧＤＮＦ）基因，并在Ｅ．ｃｏｌｉ中获得表达。方法：从人多形性成胶质细胞瘤细胞株ＢＴ３２５ 中提取总ＲＮＡ ，利用ＲＴ－ＰＣＲ技术分离ＧＤＮＦ基因，构建表达载体ｐＢＶ－ＧＤＮＦ，转化大肠杆菌，温控诱导ＧＤＮＦ的表达。结果与结论：分离得到的人ＧＤＮＦ基因，在大肠杆菌中得到高效表达，表达产物占全菌总蛋白的２４．７％ ，初步纯化后纯度达９０％ 以上。

Objective: To isolate the coding region of the glial cell line derived neurotrophic factor(GDNF) gene and to express it in E.coli. Methods: The GDNF coding sequence from the total RNA of human astrocytoma cell line BT325 was amplified. The isolated fragment was sequenced by dideoxy method, recombined in vitro with plasmid pBV220, and transformed into E.coli cells. Results and Conclusion: The GDNF coding the sequence and an E.coli strain high expressing GDNF were successfully obtained. The expressed GDNF amounted to 24.7% of the total bacterial protein. The GDNF amounted to at least 90% after purification.