Construction of pcDNA3.1(+) glial cell line derived neurotrophic factor(GDNF) vector and its expression in eukaryotic cells / 中华神经科杂志

ABSTRACT

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 cloned into pcDNA3 1(+) eukaryotic expression vector The recombinant pcDNA3 1(+)GDNF plasmid was then transfected into eukaryotic cells mediated by using Fu Gene 6 method Analysis by restricting 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 RT PCR product is 640 bp specific segment Analysis by restricting enzyme digestion and DNA sequencing of pcDNA3 1(+)GDNF recombinant showed results from restricting enzyme were 640 bp and 300 bp segments respectively DNA sequencing revealed that GDNF cloning was successful The recombinant plasmid can express active GDNF protein in eukaryotic cells Conclusion The study on the role of both GDNF and gene therapy is significant in the treatment of Parkinson disease