[Construction and identification of eukaryotic expression plasmid pcDNA3. 1-BACE and its transient expression in COS-7 cells].
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.


CONCLUSIONS
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.
Dong
Weijiang
W
Anatomy and Histology &. Embryology, Medical School of Xi'an Jiaotong University, Xi'an Shaanxi, 710061, PR China.
Gong
Huilin
H
Feng
Gaifeng
G