摘要
目的 构建 pc DNA3.1(+) GDNF真核表达质粒并了解其在真核细胞内的表达。方法 将 GDNF逆转录聚合酶链式反应 (RT- PCR)产物克隆至 pc DNA3.1(+)真核表达载体上 ,经酶切鉴定及测序分析并以 FuGene 6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 DNA3.1(+) GDNF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 GDNF蛋白。结论 以 Fu Gene 6介导 pc DNA3.1(+) GDNF质粒转染真核细胞为基因治疗帕金森氏病奠定了一定基础。
Objective To construct pcDNA3.1(+)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 pcDNA3.1(+) eukaryotic expression vector.The recombinant pcDNA3.1(+)GDNF plasmid was then transfected into eukaryotic cells mediated by Fu Gene 6.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 pcDNA3.1(+)GDNF 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.Conclusions Further study on the role of both GDNF and gene therapy is helpful in the treatment Parkinson's disease.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2001年第1期54-56,共3页
Chinese Jouranl of Endemiology
关键词
胶质细胞源性神经营养因子
基因治疗
真核细胞
帕金森病
质粒
Glial cell line-derived neurotrophic factor
Gene therapy
Eukaryotic cells
Transfection
Reverse transcription polymerase chain reaction
