大鼠GDNF真核表达载体的构建及其真在核细胞中的表达

GDNF cloning and its expression in eukaryotic cells

目的：构建ｐｃ Ｄ Ｎ Ａ３／ Ｇ Ｄ Ｎ Ｆ真核表达质粒并了解其在真核细胞内的表达． 方法：将 Ｇ Ｄ Ｎ Ｆ逆转录聚合酶链式反应产物克隆至ｐｃ Ｄ Ｎ Ａ３ 真核表达载体上，经酶切鉴定及测序分析并以脂质体介导法转染真核细胞，了解其在细胞内的表达及其表达蛋白的生物学活性． 结果：酶切鉴定及测序分析表明重组ｐｃ Ｄ Ｎ Ａ３／ Ｇ Ｄ Ｎ Ｆ表达质粒克隆成功，转染实验表明重组质粒能在真核动物细胞中表达出具有活性的 Ｇ Ｄ Ｎ Ｆ蛋白．结论：以脂质体介导ｐｃ Ｄ Ｎ Ａ３／ Ｇ Ｄ Ｎ Ｆ质粒转染真核细胞有可能用于帕金森氏症的基因治疗．

AIM: To construct pcDNA3/GDNF recombinant eukaryotic expression plasmid and to investigate its expression in eukaryotic cells. METHODS: The coding sequence of GDNF was amplified from rat astrocytes by reverse transcription PCR (RT PCR) and was cloned into pcDNA3 eukaryotic expression vector. The recombinant pcDNA3/GDNF plasmid was then transfected into eukaryotic cells mediated by transfection reagent. Analysis by restriction enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid. GDNF protein and its activity were then determined using GDNF/pcDNA3 plasmid transfected eukaryotic cells. RESULTS: The results of restriction enzyme and DNA sequencing revealed that GDNF cloning was successful. The recombinant plasmid could express active GDNF protein in the eukaryotic cells. CONCLUSION: Further study on the role of both GDNF and gene therapy is helpful in the treatment of neurodegenerative diseases.