摘要
BACKGROUND： The deposition of α -synuclein （ α -syn） aggregates is a neuropathological feature of Parkinson＇s disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson＇s disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson＇s disease. OBJECTIVE： To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING： The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University ＆ Chongqing Key Laboratory of Neurology. MATERIALS： The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS： The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS： The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION： Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.
BACKGROUND： The deposition of α -synuclein （ α -syn） aggregates is a neuropathological feature of Parkinson＇s disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson＇s disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson＇s disease. OBJECTIVE： To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING： The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University ＆ Chongqing Key Laboratory of Neurology. MATERIALS： The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS： The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS： The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION： Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.
