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[Construction and identification of eukaryotic expression plasmid pcDNA3. 1-BACE and its transient expression in
Weijiang Dong1, Huilin Gong, Gaifeng Feng
1Anatomy and Histology &. Embryology, Medical School of Xi'an Jiaotong University, Xi'an Shaanxi, 710061, PR China.
Objective:
To generate eukaryotic expression vector of pcDNA3. 1-beta-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells.
Methods:
A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1. The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger-dideoxy-mediated chain termination. The expression of the BACE gene was detected by immunocytochemistry.
Results:
The results showed that the cDNA fragment included 1.5 kb total coding region. The recombinant eukaryotic cell expression vector of pcDNA3. 1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells transfected with the pcDNA3. 1-BACE plasmid expressed a high level of the BACE protein in the cytoplasm.
Conclusion:
The recombinant plasmid pcDNA3. 1-BACE can provide a very useful tool for the research on the cause of Alzheimer's disease and lay an important foundation for preventing Alzheimer's disease.
