pEGFPN1-GDNF真核表达载体的构建及其表达

Construction of recombinant eukaryotic expression vector pEGFPN1-GDNF and its expression

目的构建人胶质细胞源性神经营养因子（GDNF）基因真核表达载体并观察其在COS-7细胞内的表达。方法应用RT—PCR从U251细胞总RNA中扩增GDNF cDNA，将其克隆至真核表达载体pEGFPN1，经酶切鉴定及序列分析后，以FugeneHD介导转染COS-7细胞，应用免疫细胞化学和Western blot鉴定其在细胞内的表达。结果RT—PCR产物为650bp的特异片段，重组质粒pEGFPN1-GDNF经双酶切产生650bp和4．7kb的片段，测序分析结果与文献报道结果完全一致。将其转染COS-7细胞后，免疫细胞化学、Western blot结果表明GDNF蛋白能在COS-7细胞中正确表达。结论成功构建了pEGFPN1-GDNF真核表达载体，为进一步开展帕金森病的基因治疗研究奠定了基础。

Objective To construct the eukaryotic expression vector pEGFPN1-GDNF and investigate its expression in eukaryotic cells. Methods The coding sequence of human glia cell line derived neurotrophic factor （GDNF） was amplified by RT-PCR from human astrocytoma cell line U251. By gene recombination technique, human GDNF coding sequence was inserted into eukaryotic expression vector pEGFPN1 that contained the reporter gene of enhanced green fluorescent protein. The recombinant plasmid was identified with restriction enzyme digestion and DNA sequencing. COS-7 cells were transfected with the recombinant plasmid by Fugene HD transfection regent. The expression of GDNF was analyzed by immunocytochemistery as well as Western blot. Results The RT-PCR product of GDNF coding sequence was 650bp specific segment. By restriction enzyme digestion, the recombinant plasmid vector was digested into 650bp and 4.7kb fragments. The immunocytochemistery and western blot showed that the GDNF was expressed successfully in COS-7 cells. Conclusion The eukaryotic expression vector pEGFPN1-GDNF was constructed successfully and the GDNF gene could be expressed successfully in eukaryotic cells. It will provide the foundation for the further research about gene therapy of Parkinsong disease.