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从人胎脑组织中提取总 RNA,以 RT- PCR方法获取编码胶质细胞源性神经营养因子 (GDNF)成熟蛋白的 c DNA。将人 GDNF c DNA插入含 T7启动子的质粒 p ET- 2 8a(+ ) ,构建表达质粒 p ET- GDNF,转化大肠杆菌获得表达菌株BL GDNF,经诱导表达的 GDNF形成包含体。凝胶自动扫描分析表明 ,表达量约占菌体总蛋白的 30 %以上。用纯化的 GDNF蛋白免疫新西兰免制备了 GDNF抗血清。纯化和复性的 GDNF蛋白能显著促进多巴胺能神经元的存活。
Total RNA was extracted from human fetal brain tissue and c DNA encoding the mature protein of glial cell line-derived neurotrophic factor (GDNF) was obtained by RT-PCR. The human GDNF c DNA was inserted into the plasmid p ET-2 8a (+) containing the T7 promoter to construct the expression plasmid p ET-GDNF, and the transformed strain was transformed into E. coli to obtain the expressed strain BL GDNF. The GDNF was induced to form inclusion bodies. Gel automated scanning analysis showed that the expression amount of about 30% of total bacterial protein. GDNF antiserum was prepared without immunization of New Zealand with the purified GDNF protein. Purified and renatured GDNF protein can significantly promote the survival of dopaminergic neurons.