pLXSN-GDNF重组载体的鉴定及其包装细胞系的建立

IDENTIFICATION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR RECOMBINANT RETROVIRAL VECTOR AND ESTABLISHMENT OF ITS PACKAGING CELL LINE PA317

目的 鉴定携带大鼠胶质细胞源性神经营养因子（glial cell line—derived neurotrophic factor，GDNF）的重组逆转录病毒载体（pLXSN—GDNF），并建立包装细胞系。方法 采用脂质体包裹法进行pLXSN—GDNF质粒PA317细胞的包装、G418抗性筛选及病毒液提取，PCR及RT—PCR进行重组逆转录病毒的鉴定，NIH3T3细胞进行逆转录病毒颗粒滴度测定。结果 pLXSN-GDNF质粒测序结果与GeneBank中序列一致，所测DNA序列位于pLXSN多克隆位点Xho I。采用pLXSN—GDNF质粒转染包装细胞PA317，获得G418抗性细胞，RT—PCR证实其培养上清含有重组逆转录病毒，采用NIH3T3细胞测定病毒滴度为10^4～10^5CFU／ml。结论 pLXSN—GDNF结构正确，GDNF基因序列完整；并成功建立了pLXSN-GDNF的包装细胞系。

Objective To identify glial cell line-derived neurotrophic factor （GDNF） recombinant retroviral vector and to establish its packaging cell line PA317. Methods PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. The recombinant retroviral particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were investigated. Results Sequencing date indicated that GDNF gene was exactly identical to the sequence in the GeneBank. PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes, and virus titers in supernatants harvested from culture media of G418 resistant transfected cells were 10^4-10^5 CFU/ml. Conclusion Packaging cell line PA317/pLXSN-GDNF was established.